Sorbonne Université - Theses.fr

Sorbonne Université

Doctoral School “Life Science Complexity”- ED515

Laboratory “Evolution of centromeres and chromosome segregation”

UMR3664 Nuclear Dynamics Unit,

Institut Curie- Centre de Recherche

CenH3-independent kinetochore assembly in

Lepidoptera requires CCAN, including CENP-T

By: Nuria Cortés Silva

PhD Dissertation

Directed by: Dr. Ines Anna Drinnenberg

Presented and defended publicly on March 9th , 2021

Jury Members:

Dr. Ines Anna Drinnenberg- Thesis director

Dr. Elaine Dunleavy-Invited member

Dr. Tatsuo Fukagawa-Rapporteur/ Examiner

Dr. Emmanuèle Mouchel-Vielh-Sorbonne University representative and President

Dr. Nikolina Sekulic- Rapporteur/ Examiner

Declaration

This PhD thesis was composed by myself. It contains data that was generated solely by me or

in a collaboration framework with others. To indicate the parts of the thesis in which data was

obtained by others, I use the pronoun "we". For the parts of my thesis that are solely my own

work, I use the pronoun "I".

Abstract

CenH3-independent kinetochore assembly in Lepidoptera

requires CCAN, including CENP-T

Chromosome segregation is an essential process to ensure the accurate transmission of DNA

during cell division. At the heart of this process are centromeres, chromosomal regions where

microtubules attach to pull the sister chromatids apart during cell division. Any alteration to

centromere function can have to severe consequences on the fitness of an organism leading

to an abnormal chromosome number (aneuploidy). In most eukaryotes, centromeres

incorporate a highly conserved centromeric-specific histone H3, CenH3 (first identified as

centromeric protein A in humans (CENP-A)), which is critical for the assembly of the

kinetochore protein complex on centromeric DNA. The kinetochore in turn fulfils the essential

function to connect and regulate spindle microtubule attachment during cell division. The

absolute requirement of CenH3 for the centromeric recruitment of all known kinetochore

components established CenH3 as the cornerstone for kinetochore assembly.

Unexpected to its conserved and essential function, it was previously shown that CenH3 was

lost independently in multiple lineages of insects; all these lineages are associated with

independent transitions from monocentricity (centromere localization is limited to one region

of the chromosomes) to holocentricity (centromeres span in large regions of the

chromosomes) (Drinnenberg et al., 2014). This discovery suggests that these insect species

employ an alternative kinetochore assembly mechanism that occur in a CenH3-independent

manner. Intriguingly, despite the loss of CenH3, previous computational predictions

performed in lepidopteran (butterflies and moths) cell lines revealed that other kinetochore

protein homologs are still present and must therefore be recruited in a different manner.

Subsequent proteomics analyses expanded this set of conserved kinetochore components

even further, and among those, identified a divergent homolog of CENP-T. In vertebrates and

fungi, CENP-T is core kinetochore component bridging centromeric DNA to the outer

kinetochore.

During my PhD I aimed to characterize the identified kinetochore components and the

assembly cascade of CenH3-independent kinetochores in Lepidoptera. In particular, I focused

on understanding the role of CENP-T in Lepidoptera. In order to address these aims, during

the first years of my PhD, I established tools (stable cell lines, antibodies) and optimized

protocols (RNAi-mediated depletion, characterization of mitotic phenotypes) in lepidopteran

cell lines as new experimental model systems. In this context, I depleted multiple kinetochore

components and studied their mitotic defects. Indeed, depletion of CENP-T and all the other

analyzed kinetochore components resulted in mitotic defects thereby supporting their

function at the CenH3-independent kinetochore. I could also show that the recruitment of

CENP-T depends on other components of the DNA proximal inner kinetochore. Furthermore,

the depletion of CENP-T and any other inner kinetochore components tested impair the

recruitment of spindle-interacting outer kinetochore portion. Using an artificial tethering

system that I developed I could also demonstrate that the presence of CENP-T is not only

necessary but also sufficient for outer kinetochore recruitment. In collaboration with the

Katsuma lab at the University of Tokyo, we could also establish an essential function of CENP-

T in vivo. Finally, the retention of CENP-T and additional kinetochore protein homologs in

other independently-derived CenH3-deficient insects indicates a conserved mechanism of

kinetochore assembly between these lineages.

Taken together, these analyses provide the first functional insights into the assembly of an

otherwise conserved kinetochore complex that functions independently of CenH3. Our results

also contribute to the emerging picture of an unexpected plasticity to build a kinetochore.

Acknowledgements

I thank my supervisor Dr Ines Anna Drinnenberg for her great scientific and personal support.

For always having her door open to have a quick chat and for considering my ideas, for her

positive attitude towards work that is also reflected on the great ambience that the lab has. I

thank you for giving me the opportunity to join your lab first as a Master student, and then to

stay at the lab as a PhD student. For always giving me the freedom to try new experiments.

Many thanks for caring about our professional and personal development as PhD students,

and for giving us the tools to accomplish our goals. In particular, thank you for being more

than a supervisor, being someone that we can count on.

I thank all the present and past members of the Drinnenberg lab for making the lab a great

place to be at. Thank you Héloïse for all the cool scientific and non-scientific chats, for your

guidance as the master of cloning :) and for being the extra moral support that I needed in

harsh times. Thanks Aruni and José for all the great moments and your words of

encouragement and advice. Jon thank you for your positive spirit and always taking care of

us. Ilham thank you for your help on protein expression and purification experiments and

always giving us a big smile. I would like to especially thank Gaétan who help me a lot carrying

out and optimizing many of the experiments of this work. Thank you for being patient with

me and always being willing to help us.

I would also like to thank all the past and present members of the Nuclear Dynamics unit

(UMR3664) for the stimulating scientific discussions and collaborative environment that you

all created. I thank the group leaders of the unit: Dr Angela Taddei, Dr Genevieve Almouzni,

Dr Antoine Coulon and Dr Nathalie Dostatni, as well as my thesis committee members for

their continuous feedback. I would particularly like to thank Dr Nathalie Dostatni for allowing

me to assist her with the organization of the international course on epigenetics. It was a

great experience and a lot of fun, I will definitely miss being involved in this event. Many

thanks to all the administrative assistants for their work and guidance. I particularly thank you

Marion for always having a big smile, while doing the impossible to help us.

Thank you all present and past members of the Fachinetti lab for your encouragement and

valuable inputs to my project during our monthly meetings. I would like to thank Dr Daniele

Fachinetti for always looking out for us.

I thank all my thesis jury members. Dr Tatsuo Fukagawa and Dr Nikolina Sekulic for their

advice and comments on the dissertation, but also for their valuable inputs on this work since

we met during the centromere meeting in 2018. Dr Emmanuèle Mouchel-Vielh who was my

professor at the Sorbonne University during my masters, the president of my jury during my

PhD entry examination and now kindly accepted the invitation of being part of my jury. Thank

you for believing in this project. I thank Dr Elaine Dunleavy for being a wonderful advisor as

part of my thesis committee and now as a member of this jury. For all the recommendation

letters and support on the next step of my scientific career.

Finally, but not less important, this work is dedicated to my family. Thank you, mom and dad,

for always supporting me, for motivating me to always have goals in life, for giving me the

greatest encouragement words and for reminding me that I will always have a home to go

back to. To my brothers who are always there when I need them, my elder brother for

showing us that there are bigger places and opportunities that we thought. Thank you, Adrian,

for being there when I was going arriving home, and specially for always protecting the people

I care the most. We will miss you. I thank my friends for the great times we shared and their

patience when I was taking about kinetochores during our weekend brunches. Many thanks

to my partner for his support, his incredible patience and for showing me that there are things

more important than work. I thank my two greatest friends who have always been on my side

making me feel at home.

Table of Contents

List of Abbreviations ......................................................................................................... 1

Introduction ...................................................................................................................... 3

The Centromere ....................................................................................................................... 5 Definition and historical background ............................................................................................................ 5 Types of centromeres .................................................................................................................................... 6

Monocentromeres .................................................................................................................................... 7 Clues towards an epigenetic determination of regional centromeres .............................................. 10

Holocentromeres .................................................................................................................................... 13

The Kinetochore ..................................................................................................................... 17 Kinetochore vs centromere ......................................................................................................................... 17 Centromeric Histone H3 (CenH3)CENP-A ........................................................................................................ 18

The Discovery of CenH3 (CENP-A) .......................................................................................................... 18 CenH3 is the epigenetic marker for the centromere function ............................................................... 20 CenH3 is the structural foundation of the kinetochore .......................................................................... 23

The kinetochore ........................................................................................................................................... 25 CCAN ............................................................................................................................................................ 25

Discovery of centromere/kinetochore components in animals and fungi ............................................. 26 CENP-B .................................................................................................................................................... 30 CENP-C .................................................................................................................................................... 32 CENP-T/W/S/X ........................................................................................................................................ 34 CENP-H/I/K/M ......................................................................................................................................... 37 CENP-L/N ................................................................................................................................................ 41 CENP-O/P/Q/U/R .................................................................................................................................... 43

Outer kinetochore ....................................................................................................................................... 47

Glimpses into kinetochore diversity ........................................................................................ 51 Kinetoplastids: complete kinetochore rewiring .......................................................................................... 53 Early divergent fungi: without CenH3 and CENP-C ...................................................................................... 55 CenH3 has been lost multiple independent times in holocentric insects ................................................... 56

Research aims ................................................................................................................. 59

Results ............................................................................................................................ 61

Discussion ....................................................................................................................... 79

Methods ......................................................................................................................... 85

Supplementary Figures ................................................................................................. 107

Bibliography ................................................................................................................. 121

Annex ........................................................................................................................... 141

1

List of Abbreviations

°C: degrees Celsius

A aa: amino acids ACA: anti-centromere autoimmune serum ATP: Adenosine triphosphate

B bp: base pair(s) BSA: bovine serum albumin

C CaCl2: calcium chloride C-banding: constitutive heterochromatin

or centromere banding CCAN: Constitutive centromere-associated

network CDC10: Saccharomyces cerevisiae gene

coding for the septin CDC10 CDE: conserved DNA elements cDNA: complementary DNA CEN: centromeric DNA CEN3: centromere of Saccharomyces

cerevisiae chromosome III CENP: centromere protein ChIP: chromatin immunoprecipitation Co-IP: co-immunoprecipitation CREST: calcinosis/Raynaud’s

phenomenon/esophageal dysmotility/sclerodactyly/telangiectasia

C-terminal/terminus: carboxyl terminal

D DAPI: 4′,6-diamidino-2-phenylindole DNA: deoxyribonucleic acid DNase I: deoxyribonuclease I DSP: dithiobis(succinimidyl propionate dsRNA: double-stranded RNA

E e.g.: exempli gratia, for example

EDTA: ethylenediaminetetraacetic acid EM: electron microscopy etc.: et cetera, and other similar things

F FANCM: Fanconi anemia

complementation group M FLAG: peptide sequence DYKDDDDK FRAP: fluorescence recovery after

photobleaching

G G-banding: Giemsa banding GFP: Green fluorescent protein

H H3: histone 3 H3S10ph: anti- phospho histone H3-Ser10 HeLa: the first immortal human cell line

identified from cervical cancer cells obtained from Henrietta Lacks in 1951

HFD: histone fold domain His: histidine HJURP: Holliday junction recognition

protein HP1: Heterochromatin protein 1

I i.e. id est "that is" ICEN: Interphase centromere complex IF: immunofluorescence IgG: immunoglobulin G

K kd: kilodalton KKT: kinetoplastid kinetochore protein KMN: KNL-1, the Mis12 and the Ndc80

complexes KNL: Kinetochore null protein KO: knockout

2

L LacI: lactose inhibitor LacO: lac operator LEU2: Saccharomyces cerevisiae gene

coding for the 3-isopropylmalate dehydrogenase

M M: molar MBP: maltose binding protein MeOH: methanol min: minute mg: milligram ml: milliliter mM: millimolar MNase: micrococcal nuclease mRNA: messenger RNA MTSB: microtubule-stabilizing buffer

N NAC: nucleosome associated complex NaCl: sodium chloride Ni: nickel nm: nanometer N-terminal/terminus: amino terminal

P PBS: phosphate buffered saline PCR: polymerase chain reaction

PFA: paraformaldehyde pg: picogram PVDF: polyvinylidene difluoride

R RNA: ribonucleic acid RNAi: RNA interference rpm: evolutions per minute

S S2 cells: Schneider 2 cells, a Drosophila

melanogaster cell line SDS: sodium dodecyl sulfate sec: second SEC: size-exclusion chromatography SID1: systemic ribonucleic acid

interference-deficient 1 SUMO: small ubiquitin-like modifier

T TAP: Tandem affinity purification TIFF: tagged image file forma Tris-HCl: tris-hydrochloride

U g: microgram l: microliter m: micrometer

3

Introduction

4

5

The Centromere

Definition and historical background

The centromere is a specialized chromosomal region that is essential for faithful transmission

of genetic material into daughter cells during cell division. This region was first observed by

light microscopy in dividing Salamander cells and hand-drawn by Walther Flemming (Figure

a) in 1882. In his drawings, Flemming

represented nuclear threads (or Mitosen as he

named) arranged in the center of the cell during

Metakinese (or metaphase) with spindle fibers

attached onto a single region (i.e. primary

constriction). Furthermore, he observed that

these threads were split longitudinally and he

predicted that one half of this longitudinally split

pair was predestinated to go to one daughter cell

while the other half should be destined to the

other daughter cell (Flemming, 1882; Paweletz,

2001). These nuclear threads were later referred

as Chromosomen (for “stainable bodies”, now

known as chromosomes) by Waldeyer in 1888

(Waldeyer, 1888).

It was in 1936 when Cyril Dean Darlington used the term “centromere” to designate the

primary constrictions or “spindle fiber attachments chromosome” (Darlington, 1936a). For

Darlington, “the centromeres are specialized chromomeres and determine chromosome

movement”, as they “arrange themselves in an equatorial plate across the spindle. They are

repelled by the centrosomes and by one another” (Darlington, 1936a). Furthermore, by

studying the meiosis in triploid plants (organisms with three homologous chromosomes), he

concluded that centromeres “modify the structural orientation of the spindle, and at

anaphase they control the spindle […]”, in these ways “the centromeres are analogous to the

centrosomes, and for this reason I have adopted the term centromere to describe

them”(Darlington, 1936b).

Figure a. Drawings of chromosome segregation in dividing

Salamander cells by Walther Flemming (Flemming, 1882).

Picture from Pawletz, 2001.

6

Decades later, studies aimed to describe the genetic nature of the centromere. In 1970, Mary

Lou Pardue and Joseph G. Gall applied hybridization techniques using radioactive nucleic acid

probes to cytologically localize specific sequences in the genome and found that sequences of

satellite DNA were located at the centromeres of mouse chromosomes. Other studies in other

organisms found that satellite DNA is also at centromeres in Rhynchosciara hollaenderi (sciarid

fly), Triturus viridescens (newt) and Drosophila melanogaster (Pardue and Gall, 1970).

Additionally to these hybridization techniques, modifications of Giemsa staining, notably G-

banding (Caspersson et al., 1970; Arrighi and Hsu, 1971; Drets and Shaw, 1971; Seabright,

1972) and C-banding (McKay, 1973) allowed the distinction between heterochromatic regions

of chromosomes (G-banding) and constitutive heterochromatin present at centromeres (C-

banding). These techniques were also widely used to characterize and karyotype

chromosomes, by for example distinguishing chromosomes based on their distribution of the

C band position.

Even though the word “centromere” from the Greek kentron for “center” and meros for “part”

(McKay, 1973), implies a position at the center of the chromosomes, this region can be located

in very different positions: in metacentric chromosomes the centromere is located in the

middle, in acrocentric chromosomes the centromere position creates a short arm and a long

arm and telocentric chromosomes are when the centromere is positioned closed to the

chromosome’s end (reviewed in Musacchio and Desai, 2017). These distinctions apply to

monocentromeres, which is one of two types of centromeric architectures that will be

described in the following section.

Types of centromeres

Monocentromeres are centromeres that are restricted to one region on each chromosome

(Figure b). This architecture is found in most eukaryotes and have first been visualized and

described as primary constrictions in Flemming’s early drawings of salamander cell mitosis

(Flemming, 1882).

7

Monocentromeres

The simplest monocentromere is the budding yeast point centromere, which consists of

unique short DNA sequences and captures a single microtubule (Peterson and Ris, 1976). The

centromeric DNA (CEN) of the budding yeast Saccharomyces cerevisiae was isolated by Clarke

and Carbon using a technique called overlap hybridization screening or chromosome

“walking” (Chinault and Carbon, 1979). In brief, they first cloned fragments containing two

Eukaryotic Centromeres

Monocentromeres

Ben E. Black University of Pennsylvania

School of Medicine

Regional centromeresPoint centromeres

S. cerevisiae stained for centromeric protein.

Erica Hildebrand, Sue Biggins lab

Holocentromeres

Bram Prevo, Arshad Desai labC. elegans stained for centromeric

protein.

Tandem repeat

(e.g. α-satellite DNA)

Specific centromere sequence

H3 nucleosomeCenH3/CENP-A

nucleosome

H3 nucleosomeCenH3/CENP-A

nucleosome

Figure b. Types of centromeres in eukaryotes. In monocentric chromosomes the centromere is restricted to one region. In

contrast in holocentric chromosomes, the centromeric regions span in large portions or even the full length of the

chromosomes. Figure adapted from McKinley and Cheeseman, 2016, and Kursel and Malik, 2016.

8

centromere III-linked genes (LEU2 and CDC10) located on opposite sides of CEN3

(chromosome III centromeric DNA) (Clarke and Carbon, 1980a). These genes were then used

to “walk” through the DNA sequence in between. The DNA sequences were also isolated using

the same overlap hybridization technique which allow them to map CEN3. Then, the isolated

sequences were reintroduced into yeast using a shuttle plasmid carrying a yeast replication

origin to test which of these sequences conferred stability to the shuttle vector over mitotic

and meiotic divisions. Using this methodology, Clarke and Carbon narrowed down the region

to a 1.6 kb segment containing the CEN3 (Clarke and Carbon, 1980a). Later studies showed

that the minimal functional budding yeast centromere (Figure c) encompass three conserved

DNA elements (CDE): CDEI (8 base pairs (bp) palindromic sequence), CDEII (78-86 bp) and

CDEIII (25 bp) giving in total a 120 bp sequence (Fitzgerald-Hayes et al., 1982; reviewed in

Pluta et al., 1995; Clarke, 1998). Among these, the 8-bp CDEI motif is also found in the

promoters of a number of genes and bound by the transcriptional activator Cpf1 (Baker et al.,

1989; Bram and Kornberg, 1987; Cai and Davis, 1990; Mellor et al., 1990). CDEIII is bound by

the point centromere-specific CBF3 protein complex which is absolutely essential for

centromere function. In fact, a single base mutation in the CCG motif of CDEIII impairs CBF3

binding thereby abolishing centromere function (Lechner and Carbon, 1991). The DNA

sequence recognized by the CBF3 protein complex contributes to the genetic identity of this

type of centromere. Thus, this sequence is both necessary and sufficient for chromosome

segregation.

Most monocentric organisms including humans (Manuelidis and Wu, 1978; reviewed in

Aldrup-Macdonald and Sullivan, 2014), Arabidopsis thaliana (Copenhaver et al., 1999) and D.

melanogaster (Sun, 2003) have “regional centromeres” that capture several microtubules and

encompass kilobases to megabases of DNA (reviewed in: Pluta et al., 1995; Muller et al., 2019)

(Figure c). In contrast to the point centromere, epigenetic factors (see next section) are crucial

for the identity and inheritance of regional centromeres. Regional centromeres are usually

composed of repetitive DNA such as satellite DNA and transposable elements (reviewed in

Muller et al., 2019). In most organisms (except for species belonging to the Candida clade

(Sanyal et al., 2004; Mishra et al., 2007)), the centromeric core of regional centromeres is

9

flanked by large regions of heterochromatin, named pericentric heterochromatin. This

common structure of regional centromeres is further detailed using the fission yeast

(Schizosaccharomyces pombe) and human centromeres as examples. (reviewed in: Pluta et

al., 1995; Muller et al., 2019).

The centromere of fission yeast is among the first regional centromeres to be characterized

(Figure c). This regional centromere spans from 40 to 100 kilobases of DNA (Fishel et al.,

1988). It consists of a non-repetitive DNA central core region (cnt) flanked by innermost (imr)

and outermost (otr) repetitive DNA (Clarke et al., 1986; Chikashige et al., 1989; Clarke and

Baum, 1990; Hahnenberger et al., 1991; Murakami et al., 1991; Steiner et al., 1993). The

central core region functions as binding site for the kinetochore-specific proteins (see chapter

2) (Polizzi and Clarke, 1991; Thakur et al., 2015), while the flanking repeat region recruit

cohesion to enable tight association between sister centromeres.

Human centromeric core DNA encompasses A-T rich alpha-satellite repeats (Jones and

Corneo, 1971; Jones et al., 1974; Manuelidis, 1978a, 1978b) based on 171 bp monomer

repeat unit arranged in a head-to-tail fashion (Manuelidis and Wu, 1978; Willard, 1985;

Willard and Waye, 1987) (Figure c). Subsequent studies showed that these monomers

Figure c. Centromere structure in different organisms. Centromeres in eukaryotes are highly diverse in DNA composition, size

and position. Figure adapted from Muller et al., 2019 showing the typical centromere organization for model organisms. When

a specific chromosome is represented, its number is indicated. The approximate scale is shown on the right.

10

assemble into a higher-order-repeat pattern (HOR), which are up to > 90% identical to each

other at the center of the satellite repeat array (Waye and Willard, 1989). Flanking the HOR

array, which is bound by kinetochore-specific proteins, monomers are more randomly

arranged. The centromeric DNA length varies between chromosomes, creating chromosome-

specific arrays of 0.25-5 megabases and representing 3-5% of the chromosome (Willard, 1985;

Choo et al., 1991; reviewed in: Aldrup-Macdonald and Sullivan, 2014; Fukagawa and

Earnshaw, 2014; Kursel and Malik, 2016). Due to high sequence similarities within the satellite

repeat arrays, it has been challenging to assemble and characterize the HOR structures of

human centromeres. Nevertheless, more recent efforts using long-read sequencing

completed centromeric assemblies for chromosomes 8, X and Y (Schueler et al., 2001;

Nusbaum et al., 2006; Miga et al., 2014; Jain et al., 2018).

Note, that repetitive DNA is not common to all regional centromeres. Some organisms

including chickens (Shang et al., 2010), horses (Piras et al., 2010) or orangutans (Locke et al.,

2011) also contain chromosomes with non-repetitive regional centromeres, which has been

hypothesized to represent an evolutionary more recent, immature state (Nergadze et al.,

2018).

Clues towards an epigenetic determination of regional centromeres

As described above, the centromere localization in point centromeres is dependent on the

120 bp centromeric sequence. This conclusion comes from experiments showing that these

sequences are sufficient to confer mitotic and meiotic stability when introduced to a shuttle

plasmid, creating a mini chromosome in budding yeast (Clarke and Carbon, 1980b). In

contrast, in other monocentric organisms with regional centromeres, the centromeric

function does not depend on specific DNA sequences (reviewed in: McKinley and Cheeseman,

2016; Murillo-Pineda and Jansen, 2020).

One of the first evidences to support that regional centromeres are not genetically defined

came from human samples with stable dicentric X chromosomes (X chromosomes with two

11

centromeres). It was proposed that these dicentric

chromosomes were stable because one of the two

centromeric regions was inactive. This results in dicentric

chromosomes that functionally behaved as

monocentromeres. Indeed, it was shown that centromere-

associated proteins were absent in the inactive centromere

(Earnshaw and Migeon, 1985) (Figure d) regardless of the

presence of -satellite DNA (Warburton et al., 1997).

Taken together, these results suggested that the

centromere identity in regional centromeres is not defined

by specific sequences, rather centromere-associated

proteins act has an epigenetic mark specifying active

centromeres localization. Similarly, subsequent work in S.

pombe described that centromere function of centromeric

DNA cloned into yeast artificial chromosomes was under

an epigenetic mechanism, which allowed the activation of

a nonfunctional centromere into a functional centromere

without changes in sequence or structural arrangement of

the minichromosomal DNA (Steiner and Clarke, 1994).

Indeed, recent studies have shown that S. pombe cells with

dicentric chromosomes survive when dicentric

chromosomes convert into stable monocentromeres via an

epigenetic centromere inactivation mechanism. This

mechanism involves disassembly of the microtubule-

binding machinery, heretochromatinization and histone

deacetylation expanding from pericentric repeats to the central domain. Together these

changes prevent the reactivation of the inactivated centromere (Higgins et al., 2005; Sato et

al., 2012). A similar mechanism has been also described in centromere inactivation in human

centromeres (Higgins et al., 2005). These data together led to the notion that the centromere

sequences were not sufficient to define the centromere function and pointed to the idea that

centromere function is rather defined epigenetically (Sullivan et al., 2001).

Figure d. Centromere proteins are absent

at inactive centromeres. a) Mitotic

chromosomes stained with the anti-

centromere antibody. b) Superimposition

of phase-contrast and fluorescent images

showing the chromosomal localization of

centromeric proteins (white dots). c) Q-

banding staining the entire chromosome

lengths. The large arrow indicates the

dominant centromere on the dicentric

chromosome and the small arrow indicates

the inactive centromere. Scale bar: 10 m.

Figure from Earnshaw and Migeon, 1985.

12

In addition, neocentromeres also provided evidence to the notion that centromere sequences

are neither necessary for the centromeric function. In 1993, a karyotype analysis of a human

patient sample revealed the absence of a normal chromosome 10. Instead the patient had

two chromosomal fragments corresponding to this chromosome (as their aggregate size was

approximately the size of a normal chromosome 10). Cytological analysis of the sample,

suggested that there were no translocations or fragments of other chromosomes implicated

into creating these fragments. Furthermore, one of these chromosome 10 fragments was able

to segregate as it was present in the majority of daughter cells after cell division. Nevertheless,

the most intriguing observation was that this stable fragment showed no detectable

centromeric heterochromatin signal, while it showed enrichment for microtubule-binding

proteins explaining its capacity to segregate. This data suggested for the first time that alpha-

satellite centromeric sequences were not necessary for centromere function (Voullaire et al.,

1993).

Similar conclusions resulted from studies in other human chromosomes. For instance, in a

patient sample, the Y chromosome showed two centromeric regions with only one located at

the usual primary construction. Intriguingly, the neocentromere was enriched for

microtubule-binding proteins (CENP-A and CENP-C, further discussed in the next chapter)

while the satellite DNA sequence signal was only localized at the usual centromere position

(Tyler-Smith et al., 1999). The formation of stable neocentromeres in humans without any

apparent enrichment on centromeric DNA or even no apparent chromosomal rearrangement

(Amor et al., 2004) has widely been described (reviewed in: Marshall et al., 2008; Murillo-

Pineda and Jansen, 2020).

Efforts have also been made to generate and study neocentromeres in other organisms. For

example, in D. melanogaster gamma irradiation of chromosome X resulted in acentric mini

chromosomes. These mini chromosomes were devoid of centromeric DNA and were able to

self-propagate by acquiring centromere function without integration of any centromeric DNA

sequence (Williams et al., 1998). Similarly, chromosome engineering methods in chicken DT40

cells allow the creation of neocentromeres (Shang et al., 2013) to understand their formation

and specification.

13

Though centromere specification seems to be strongly based on epigenetic mechanisms,

genetic features may also contribute to the centromere identity and function. Many efforts

are made in the centromere field to generate neocentromeres in different organisms which

will allow to define features and requirements for centromere formation.

Holocentromeres

In contrast to the monocentric architecture, several organisms have holocentromeres in

which the centromeric microtubule-binding regions span in large portions or even the full

length of the chromosomes (Figure b). This type of centromeric architecture was first

described by Franz Schrader based on cytological analyses of chromosome segregation in

Hemiptera (true bugs) during cell division. From his observations, he concluded that “in

Hemiptera […] the entire pole-ward surface of the chromosome serves as a base for the half

spindle component” (Schrader, 1935).

Since then the criteria used to characterize holocentric chromosomes has been based on

cytological remarks (Hughes-Schrader and Ris, 1941).

These include (Figure e):

i) the lack a primary constriction,

ii) migration pattern and sister chromatids shape during cell division. Indeed, during

anaphase the sister chromatids migrate to the spindle poles as parallel lines (since

microtubule-pulling forces are distributed along the chromosomes), while

monocentromeres adopt a “V”-shape because the pulling forces are located at one

chromosomal region.

iii) Their ability to recover from chromosomal breaks. It has been thought that

holocentromeres have a greater ability to recover from chromosomal break than

monocentromeres. This is due to the fact that chromosomal fragments resulting

from DNA breakage have a high probability to possess at least one centromeric site

and therefore to segregate (reviwed in: Maddox et al., 2004; Mandrioli and

Manicardi, 2020).

14

Holocentricity is not a rare event during evolution.

Instead it appears to have evolved at least 13

independent times in animals and plants (Melters

et al., 2012). However as cytologically-based

methods are the main driver to identify holocentric

species, it is possible that the prevalence of

holocentromeres is underestimated as there are

many species that are difficult to study

cytologically.

The holocentric organism best characterized is

worm C. elegans. In this nematode,

immunostaining of the centromere-specific protein

(CenH3HCP-3 see chapter 2) displays a localization as

a longitudinal band along the entire length of the

chromosome (Buchwitz et al., 1999). A genome-

wide study using ChIP-microarray mapping of C.

elegans embryos revealed that CenH3 incorporates

at low abundance into larger domains of complex

DNA that encompass half of the genome.

Moreover, the authors found that those

centromeric domains are enriched over

transcriptionally silent regions leading to the

hypothesis that centromere formation requires

low chromatin activity (Gassmann et al., 2012).

Another later study mapped the centromere sites

in C. elegans embryos using native ChIP followed by

sequencing. Here similarly to Gassmann et al., the authors found that C. elegans CenH3

incorporates at low abundance into large domains along the chromosomes. Moreover, the

authors also found that CenH3 localizes at discrete and dispersed loci with high abundance.

From their data, the authors conclude that the actual centromeric sites resemble point

centromeres as those in budding yeasts (Steiner and Henikoff, 2014). Up to date, it is still

Figure e. Cytological criteria to characterize

holocentric chromosomes. Top panel) In monocentric

chromosomes, the centromere (red circle) is located

at the chromosomal primary constriction during

metaphase (M). At anaphase (A) chromatids move

towards poles adopting “V”-shape structures. In

holocentric chromosomes, the centromere (red line) is

located along the entire length of the chromosome.

No primary constriction is present during metaphase

(M). During anaphase (A) holocentric chromatids

move towards poles as two parallel linear bars.

Bottom panel) Upon chromosomal breakage in

monocentric chromosomes, the resulting acentric

chromosome fragments would not attach to

microtubules during metaphase (M). Thus, they are

lost during anaphase (A). In contrast, upon

chromosome breakage in holocentric chromosome,

the resulting chromosomal fragments would attach to

microtubules due to the chromosome-wide

centromere extension. Therefore, these fragments

would be inherited. Figure adapted from Mandrioli

and Manicard, 2020.

15

controversial to what extend the discrete sites or larger domains contribute to centromere

function in C. elegans. In contrast to C. elegans, IF-FISH and ChIP-seq studies in holocentric

plant Rhynchospora pubera (beak-sedge) revealed that centromeric sites are located in

repeat-rich regions. The authors found that centromeres are enriched by tandem repeats and

retroelements, and are distributed as genome-wide interspersed arrays (Marques et al.,

2015). To what extend those repeats play an active role in recruiting centromeric proteins is

still unclear.

16

17

The Kinetochore

Kinetochore vs centromere

The term “kinetochore” was coined more than 90 years ago but for a long time involved in a

terminological controversy. In 1934, Lester W. Sharp (inspired by a suggestion of J.A. Moore)

used for the first time the term “kinetochore (= movement place)” to designate the “fiber-

attachment point, […] primary constriction, kinetic constriction, […]” in order to reflect the

role of this region in chromosome movements (Sharp, 1934). A few years later in 1939, Franz

Schrader also proposed to use the word “kinetochore” instead of “centromere” (Darlington,

1936a) in a letter to Nature as he felt that this word was creating confusion within the scientific

community because many other words (like “centrosphere”, “centrodesmus”, “centrosome”,

“centriole”, “central body”) were quite similar. In addition, he argued that the word

“centromere” was already coined by Waldeyer in 1903 to refer to the neck-region of the

sperm (Schrader, 1939). Despite the efforts to get into a consensus, both terms “centromere”

and “kinetochore” were used as synonyms for many years (Battaglia, 2003). It was not until

1982 when Conly L. Rieder suggested “to eliminate this confusion, I suggest that the term

kinetochore be used as defined by Ris and Witt (Ris and Witt, 1981) to note, at the

ultrastructural level, the precise region on the chromosome that becomes attached to spindle

microtubules. In mammalian cells this region differentiates into a trilaminar disk structure […],

I suggest that the term centromere be used […] to note the region on the chromosome (e.g.,

the primary constriction, peri-centromeric heterochromatin, etc.) with which the kinetochore

is associated” (Rieder, 1982).

Early visualization of the kinetochore by electron microscopy (EM) allowed to distinguish

between the “kinetochore” and the “centromere”. These studies described the complex as a

multilayered structure where microtubules attach (Harris and Mazia, 1962; Nebel and Coulon,

1962; Rieder, 1982; Robbins and Gonatas, 1964; George et al., 1965; Krishan and Buck, 1965;

Luykx, 1965; Brinkley and Stubblefield, 1966). For instance, using fertilized eggs of the Urechis

caupo (marine echiuroid worm) combined with EM, Luykx described the kinetochore as three

layers : two electron dense layers separated by an electron-transparent layer (Luykx, 1965)

(Figure f). The outer plate was calculated to be approximately from 30 to 40 nm wide, the

18

middle layer from 15 to 35 nm wide and the inner

plate of 20 to 40 nm (reviewed in Rieder, 1982).

More general, EM analyses of several laboratories

revealed that the inner plate, also called inner

kinetochore, connects to the centromeric DNA

while the outer plate, or outer kinetochore,

connects to the spindle microtubules (Figure f)

(reviewed in Musacchio and Desai, 2017).

While these studies provided the first insights into

the overall organization of the kinetochore as a

whole, the molecular composition of the

kinetochore remained unknown until the early

2000s when mass spectrometry-based proteomic

and functional genomics allowed the identification

of kinetochore subunits, their expression,

reconstitution and purification (reviewed in

Samejima et al., 2017) (further discussed in

following chapters). These molecular and

biochemical analyses described the multilayered

kinetochore complex as network of many protein

subunits that act in a coordinated manner to

ensure the dynamic assembly and function of the

kinetochore (Cleveland et al., 2003). In the

following sections, I will give an overview on the composition and role of each of the

kinetochore protein subunits, as well as their connectivity within the complex.

Centromeric Histone H3 (CenH3)CENP-A

The Discovery of CenH3 (CENP-A)

The discovery of centromere associated proteins including CenH3/CENP-A hinged

serendipitously on observations made in the 1980s. In 1980, Moroi et al., discovered an

Figure f. The kinetochore is a multilayered structure.

Top) Electron micrograph of marine echiuroid worm

chromosome in anaphase. Section through two

adjacent sister kinetochores. Two electron-dense

layers are visible (inner and outer kinetochore). These

layers are separated by an electron-transparent layer.

x 44,000. Figure adapted from Luykx, 1965. Bottom)

Mitotic chromosome sectioned along the spindle axis

plate. Key kinetochore elements are colored: inner

kinetochore (in violet), regulatory proteins (e.g.

Aurora B, in red), outer kinetochore (in yellow) and

spindle microtubules (in green). Figure adapted from

Cleveland, et al., 2003)

19

antibody localizing to centromeric regions (or primary constrictions) in a sera from patients

with the CREST scleroderma variant (Figure g-A). Indeed, autoimmune sera staining appeared

as two small dots at centromeric regions during metaphase, while it appeared as speckles

during interphase both in human, mice and Chinese hamster cells. This observations led to the

notion that this particular antibody could be recognizing a protein or proteins tightly bound

to DNA at the centromere (Moroi et al., 1980).

Figure g. A) Chromosome spread stained with sera from patients with the CREST scleroderma variant. The immunofluorescent

staining localizes to the centromeric region of the chromosomes and “each centromeric region showed two small spheres of staining resembling kinetochores” (Moroi et al., 1980). x 800. Figure adapted from Moroi et al., 1980. B) Immunoblotting

analysis of the anti-centromere sera from CREST patients. The antigen was isolated from HeLa chromosomes. Lane a:

coomassie-blue-stained gel. Lane b: whole serum. Lane c: anti-140 kd fraction (1:22) (CENP-C). Lane d: anti-80 kd fraction

(1:22) (CENP-B) and lane e: anti-20-25 kd fraction (1:22) (CENP-A). Figure adapted from Earnshaw and Rothfield, 1985. C)

CENP-A is a distinctive centromere-specific histone. “Some CENP-A sequences are highly similar to H3 regions, while others

are unrelated to H3 or any other histone” (Palmer et al., 1991). Table 1 from Palmer et al., 1991.

Further immunofluorescence and immunoelectron microscopy analyses revealed that the

CREST antiserum was specific for proteins localizing to the kinetochore complex at

centromeres (Brenner et al., 1981). Yet, the molecular composition of this antiserum

remained unclear until the mid 1980s when it was shown that this sera recognizes a 19.5

kilodalton (kd) polypeptide (Guldner et al., 1984). Indeed, immunoblotting experiments using

chromosomes from HeLa cells revealed that this antiserum recognizes three antigens. These

A)

C)

B)

20

antigens were identified as centromere components and named CENtromere Protein (CENP)

-A (17 kD), CENP-B (80 kD) and CENP-C (140 kD) (Earnshaw and Rothfield, 1985) (Figure g-B).

Subsequent studies showed that the identified centromere proteins were tightly bound to

mononucleosomes (Palmer and Margolis, 1985) and concluded based on its similar

biochemical properties and protein sequence relationship with histone H3 that CENP-A is a

histone (Palmer et al., 1991). Yet, while part of CENP-A’s amino acid sequence was similar to

that of H3, it was realized that CENP-A also contained segments that were different to those

found in other core histones (Palmer et al., 1991) (Figure g-C). Later on, sequence analyses of

full-length human CENP-A cDNA further confirmed that indeed this protein is a centromere-

specific variant of histone H3 (CenH3). It was found that CENP-A has a unique amino-terminal

and a carboxyl-domain which shares 62% identity to that of H3. Unexpectedly for this period

of time, it was C-terminal histone fold domain (HFD) and not the divergent N-terminal domain

that was responsible for CENP-A DNA-binding properties (Sullivan et al., 1994).

Homologs of CENP-A have been identified at centromeres in most eukaryotes, including

protists (Dawson et al., 2007) , budding yeast (Stoler et al., 1995), fission yeast (Takahashi et

al., 2000), flowering plants (Talbert et al., 2002), flies (Henikoff et al., 2000; Blower and

Karpen, 2001) and worms (Buchwitz et al., 1999). In addition, studies in multiple organisms

have shown that the inactivation or depletion of their respective CENP-A homologs results in

chromosome mis-segregation leading to lethality (Stoler et al., 1995; Buchwitz et al., 1999;

Howman et al., 2000; Blower and Karpen, 2001; Talbert et al., 2002).

In the following sections I will outline the two essential functions of CENP-A/CenH3 for

centromere identity and kinetochore assembly. I will use the term centromeric histone H3

variant or CenH3 when referring to this class of histone variants, with the species-specific

name of CenH3 (i.e. CENP-A, Cid, ...) added as superscript.

CenH3 is the epigenetic marker for the centromere function

As described in the previous section, early observation made using human dicentric

chromosomes led to the notion that human centromeres in contrast to the budding yeast

point centromeres are not defined by the underlying DNA sequence but instead by the

21

presence of CenH3CENP-A. This was based on the observation that CENP staining on isodicentric

X chromosomes with one active and one inactive centromere was only present at the active

centromere, despite the presence of repetitive alpha-satellite DNA at both active and inactive

centromeres (Earnshaw and Migeon, 1985) (Figure d) . A subsequent study using an antibody

specific to CENP-A for the first time, revealed that indeed CenH3CENP-A was only detected in

the inner kinetochore plate at the active centromere (Warburton et al., 1997). Studies of

human neocentromeres mainly derived from patient samples supported this notion that

alpha-satellite sequences are indeed not necessary for centromere function (Voullaire et al.,

1993; Tyler-Smith et al., 1999; Amor et al., 2004; reviewed in: Marshall et al., 2008; Murillo-

Pineda and Jansen, 2020).

More recent studies based on artificial kinetochore assembly assays using the Lac operator

(LacO) arrays /LacI system further demonstrated CenH3’s role as epigenetic marker of

centromeres. In brief, LacI-fused kinetochore proteins are artificially tethered to arrays of

LacO repeats that are located at non-centromeric chromosomal regions. Artificial tethering of

GFP-LacI tagged D. melanogaster CenH3CID (CenH3CID-GFP-

LacI) to ectopic LacO sites in S2 cells resulted in the

formation of the kinetochore at those sites. Following the

loss of the plasmid coding for CenH3CID-GFP-LacI tagged

protein, untagged CenH3CID signal was detected at the

ectopic LacO arrays (Figure h). Thus, the initial targeting of

CenH3CID-GFP-LacI protein was sufficient to recruit

endogenous CenH3 that self-propagates to inherit

centromere function (Mendiburo et al., 2011). Using the

same LacO-LacI tethering system, induced de novo

centromeres were shown to be even propagated during

Drosophila development in vivo (Palladino et al., 2020).

A sufficiency of CenH3CENP-A to assemble centromeres at

ectopic sites on human chromosomes is slightly

controversial (Barnhart et al., 2011; Gascoigne et al., 2011).

Nevertheless, multiple studies have contributed to characterize the self-propagating loop that

Figure h. Artificially tethered CenH3CID to

ectopic LacO arrays is sufficient for

centromere formation. Mitotic

chromosome expressing CID-GFP-LacI

(green) and low levels of CID-HA (red).

Top) At day 1, upon targeting of CID-

GFP-LacI to the ectopic lacO sites (green

arrows), there is only a low CenH3CID-

HA recruitment to LacO arrays and it

mostly localizes at the endogenous

centromeres (white arrows). Bottom) At

day 7, CID-HA recruitment to the

ectopic lacO sites increases. Scale

bars: 3 m Figure adapted from

Mendiburo et al.,2011.

22

involves CenH3CENP-A together with its dedicated chaperone HJURP (Holliday junction

recognition protein) (Dunleavy et al., 2009; Foltz et al., 2009) and other kinetochore

components (Fujita et al., 2007; Maddox et al., 2007; Carroll et al., 2010; Barnhart et al., 2011;

Moree et al., 2011; Dambacher et al., 2012; Kato et al., 2013; Hayashi et al., 2014; Wang et

al., 2014; Nardi et al., 2016; Stellfox et al., 2016). In contrast to human cell lines, CenH3CENP-A

appears to be sufficient for centromere formation in chicken DT40 cells as shown using the

same LacO-LacI tethering approach. Here, following removal of the endogenous centromere,

tethering of HJURP and kinetochore proteins CENP-C and CENP-I LacI fusion proteins resulted

in CENP-A recruitment at the LacO arrays and kinetochore formation (Figure i-A, B and C).

Importantly after IPTG-mediated disruption of LacI fusion protein binding to LacO arrays, the

artificial centromeres were stably inherited (Hori et al., 2013) (Figure i-D) . These observations

showed that CenH3 is sufficient to target its own incorporation thereby maintaining the

centromere function at the very same location.

Figure i. Ectopic localization of CENP-C, CENP-I and HJURP results in kinetochore formation at a noncentromere locus. A)

Experimental strategy. Following the removal of endogenous centromere of chicken cells chromosome Z (Zcen), GFP-LacI-

tagged proteins are artificially tethered to a noncentromere site. B) Localization of GFP-LacI-tagged proteins: CENP-C, CENP-I

and HUJRP before removal of endogenous centromere. The LacO locus is indicated with the white arrows. C) Immunostainings

of kinetochore proteins (in red, and indicated with the white arrows): CENP-A, CENP-C, CENP-T and Ndc80 at LacO locus in

CENP-C-LacI (CC-LacI), CENP-I-LacI (CI-LacI) and HJURP-LacI (HJ-LacI) cells. The chromosome Z was identified using the Z-

specific satellite probes (in green). D) Representative image of chromosome Z identified by the Z-specific probe (in red) in

CENP-C-LacI (CC-LacI), CENP-I-LacI (CI-LacI) and HJURP-LacI (HJ-LacI) cells after addition of IPTG. Figures adapted from Hori et

al., 2013

A)

C)

B)

D)

23

A retention of CenH3 at centromeres also contributes to the inheritance of the locus through

the germline and into the next generation. Early work showed that CenH3CENP-A is retained in

mature bovine sperm while somatic and testis specific histones are replaced by protamines

(Palmer et al., 1990). This observation suggested a possible involvement of CenH3CENP-A in the

epigenetic inheritance of centromere locus. In agreement with CenH3 role as marker for the

epigenetic centromere identity, once incorporated into chromatin, CenH3 is a long lived

protein (Shelby et al., 2000). CenH3 remains stable over several mitotic generations and its

levels only slightly diminish over time in resting oocytes (Régnier et al., 2005; Jansen et al.,

2007; Swartz et al., 2019). Taken together, these features constitute the function of CenH3 as

epigenetic marker of the centromere.

Nevertheless, additional studies also proposed that other features of CenH3-containing

chromatin are important for centromere specification (reviewed in Barrey and Heun, 2017).

CenH3 is the structural foundation of the kinetochore

In addition to epigenetically marking centromere location, CenH3 is required for the

recruitment of all known kinetochore proteins, indicating that it is the core player for defining

the site of a functional kinetochore (Cheeseman, 2014). In some organisms, CenH3 is even

sufficient for kinetochore assembly. Similar to the study in S2 cells using LacO arrays described

above, it has been shown that overexpression of CenH3CID in D. melanogaster S2 cells results

in CenH3CID mislocalization in chromosomal regions other than the centromere. CenH3CID

mislocalization then promoted formation of functional kinetochores capable of interacting

with spindle microtubules (Heun et al., 2006). In contrast, while CenH3CENP-A overexpression

in human cells also results in its incorporation at ectopic sites, this only lead to the mis-

localization of a subset of kinetochore proteins but not full kinetochore assembly (Van Hooser

et al., 2001; Gascoigne et al., 2011) (Figure j). Thus, in humans, CenH3CENP-A is not sufficient

for full kinetochore assembly. Nevertheless, it was proposed that the specialized CenH3CENP-A

chromatin structure might generate an environment that is permissive for kinetochore

assembly (Gascoigne et al., 2011).

24

Figure j. Mistargeting of CenH3CENP-A in human cells does not result in ectopic kinetochore formation. Top) Mislocalization of

CenH3CENP-A by overexpression results in corecruitment of only three kinetochore proteins (CENP-C, CENP-N and Mis18) out of

the 16 kinetochore proteins tested. Bottom) Representative pictures of kinetochore proteins (CENP-T, CENP-H and Ndc80Hec1)

that do not mislocalized upon CenH3CENP-A overexpression. Scale bars: 5 m. Figures adapted from Gascoigne et al., 2011

A) B)

25

The kinetochore

The kinetochore is a multi-protein complex that is comprised of a centromere-proximal inner

portion (inner kinetochore) and a centromere-distal outer portion (outer kinetochore).

Components of the inner kinetochore make contacts with centromeric DNA, whereas

components of the outer kinetochore interact with spindle microtubules. Assembly of the

former precedes that of the latter where the inner complex assembles on centromeric

chromatin during the course of interphase while the outer complex gets recruited upon entry

to mitosis. Taken together, the two bridge the gap between centromere and spindle during

cell division to drive chromosome segregation (reviewed in Cheeseman, 2014).

CCAN

In many organisms including vertebrates and fungi, the inner kinetochore is composed of a

complex network of up to sixteen proteins that constitutively localize to centromeres

throughout the cell cycle. This group is named

constitutive centromere-associated network (CCAN).

The CCAN can be divided up into different subfunctional

complexes: CENP-C, the CENP-H/I/K/M, the CENP-L/N,

the CENP-T/W/S/X and the CENP-O/P/Q/R/U complexes

(reviewed in Hara and Fukagawa, 2017). The CCAN is

assembled on CenH3-containing chromatin and capable

to recruit the outer kinetochore or KMN network (for

Knl1, Mis12 and Ndc80 complexes, further discussed

below) to enable the attachment to spindle

microtubules (Figure k) (reviewed in Cheeseman, 2014;

Nagpal and Fukagawa, 2016; Musacchio and Desai,

2017).

The discovery of many kinetochore components

including the CCAN was enabled using large scale

genetic screens and biochemical isolation combined

with mass spectrometric analyses of centromere

Figure k. Schematic model of vertebrate

kinetochore. CenH3CENP-A is the most upstream

component of the kinetochore and the marker

for the recruitment of the constitutive

centromere-associated network (CCAN). The

CCAN consists of CENP-C, CENP-T-W-S-X, CENP-

N-L and CENP-O-P-Q-R-U. CenH3CENP-A, CENP-C,

CENP-N and CENP-T-W-S-X have DNA-binding

properties. The outer kinetochore (composed by

the Mis12 complex, Ndc80 complex and KNL1)

is in direct contact with spindle microtubules

(green and grey spheres). Figure adapted from

Nagpal and Fukagawa, 2016.

26

associated complexes. These studies have been conducted during the last 30 years.

In the next section, I will give a brief overview of the main methods used to discover and clone

kinetochore components.

Discovery of centromere/kinetochore components in animals and fungi

As described in the previous section, the first kinetochore proteins identified were CENP-

A/B/C in humans thanks to the unexpected discovery of antibodies present in CREST patients

that reacted with the centromere (Earnshaw and Rothfield, 1985). A few years later,

biochemical purification led to the discovery of a 240 kb protein complex called CBF3 bound

to budding yeast centromeric DNA. This complex was reported to be composed by three

proteins: CBF3A (110 kd), CBF3B (64 kd) and CBF3C (58 kd) together specifically bind to CDEIII

consensus sequence, as a single base substitution within CDEIII is sufficient to abolish CBF3

binding (Lechner and Carbon, 1991). Subsequent genetic screens for genes that impact

chromosome segregation efficiency in budding yeast allowed the identification of additional

kinetochore components including budding yeast homolog CenH3, Cse4 (Stoler et al., 1995).

Similar genetic screens were also performed in the fission yeast S. pombe and identified

several outer kinetochore components and those involved in CenH3 loading (Goshima et al.,

1999; Hayashi et al., 2014; Samejima et al., 2017). While in the early 2000s most yeast

kinetochore proteins were identified, (reviewed in Biggins, 2013) this was not the case for the

vertebrate kinetochore. In fact only two vertebrate kinetochore proteins (CENP-I (Nishihashi

et al., 2002) and Mis12 (Goshima et al., 1999)) were identified based on sequence homology

to their fission yeast counterpart (reviewed in Samejima et al., 2017).

Large scale genetic screens using RNA interference (RNAi)-mediated depletions also led to the

identification of kinetochore proteins in C. elegans. Here, the first kinetochore components

identified were the C. elegans CenH3 homolog (HCP-3) and CENP-C. Depletion of these

components results in “kinetochore null” phenotype characterized by a failure of metaphase

plate formation, absence of chromosome segregation and a failure to recruit other

kinetochore components for stable binding to spindle microtubules (Oegema et al., 2001)

(Figure l).

27

Subsequent RNAi screens identified KNL-1

as a protein recruited downstream of

CenH3HCP-3 and CENP-C and necessary to

generate the microtubule-binding

interface (Desai et al., 2003). Co-

immunoprecipitation (Co-IP) of KNL-1 and

KNL-3, another kinetochore protein

identified, followed by mass spectrometry

analyses revealed a complex of ten

proteins that copurified together. Their

individual depletions resulted in similar

phenotypes, which supported that these

proteins were indeed forming a network.

This complex called the KMN network

(further discussed below) is formed by three subcomplexes: KNL-1, the Mis12 and the Ndc80

complexes, and together function as the microtubule-binding site of the kinetochore

(Cheeseman et al., 2004, 2006).

Co-IP experiments followed by mass spectrometry also allowed the identification of most

CenH3-interacting proteins in vertebrates. One of the first studies performed chromatin

immunoprecipitation (ChIP) with monoclonal CenH3CENP-A antibody in HeLa interphase nuclei

and identified 40 proteins, that together composed the CenH3CENP-A chromatin complex or

interphase centromere (ICEN) complex. Mass spectrometric analyses recovered all

kinetochore proteins reported at the time (CENP-A/B/C/H/I and Mis12) plus other proteins of

unknown function. In addition to kinetochore proteins, this analysis also identified Polycomb

group proteins and chromatin remodelers enriched in the centromeric fraction. This

observation led the authors suggest that the centromere complex might regulate

heterochromatin formation in and around the centromere (Obuse et al., 2004). Two years

later in 2006, in a follow-up paper, the authors characterized seven (ICEN22, 24, 32, 33, 36, 37

and 39) of these unknown kinetochore proteins further using cell biological methods. The GFP-

tagged proteins colocalized to CenH3CENP-A at the centromeres in interphase and in

metaphase. Furthermore RNAi-mediated depletion of all analyzed proteins resulted in

Figure l. Depletion of CenH3HCP-3and CENP-C in C. elegans

result in failure of metaphase plate formation and absence of

chromosome segregation. Panels summarizing videos of

embryos expressing GFP-histone in wild-type embryo (left), C.

elegans CenH3CENP-A-depleted embryo (CeCENP-A, middle)

and C. elegans CENP-C-depleted embryos (CeCENP-C, right) In

CeCENP-A– or CeCENP-C–depleted embryos, chromosomes

failed to form the metaphase plate and did not segregate at

anaphase. Scale bars: 5 m. Figure adapted from Oegema et

al., 2001.

28

misaligned chromosomes and aneuploidy supporting their role at the kinetochore. Among the

analyzed proteins, depletion of ICEN22, 32, 33, 37 and 39 resulted in absence of CENP-H and

CENP-I at the centromeres, while CenH3CENP-A and CENP-C continued to be present (Izuta et

al., 2006). The seven ICEN proteins were later identified as CENP-T (ICEN22), CENP-U (ICEN24),

CENP-N (ICEN32), CENP-L (ICEN33), CENP-O (ICEN36), CENP-K (ICEN37) and CENP-M (ICEN39)

(these proteins were also identified in 2006 by Foltz et al. and Okada et al., see here below)

(reviewed in Samejima et al., 2017).

Likewise, in 2006 a study using

multiple tandem affinity purification

(TAPs) defined the most proximal

complex to CenH3CENP-A in HeLa cells.

The purified CenH3CENP-A-TAP tagged

complex included known centromere

proteins CENP-B/C/H/U and three

additional proteins CENP-T, CENP-N

and CENP-M, all which were

previously identified (Obuse et al.,

2004). Their participation in the

complex was validated by reciprocal

affinity purifications targeting CENP-T, CENP-N and CENP-M. This observation suggested that

these proteins together with CENP-C and CENP-H constituted the CenH3CENP-A proximal

nucleosome associated complex (CenH3CENP-A NAC). CENP-H, CENP-N and CENP-M localization

to centromeres were abolished upon depletion of CenH3CENP-A, while CENP-B remained at

these sites. Furthermore, double affinity purification of CENP-M/N/U-TAP tagged complexes

revealed six additional components (CENP-I/K/L/O/P/Q) and two more proteins (CENP-R/S)

only present in CENP-M and CENP-U complexes (Figure m). All the identified proteins were

bound to the centromere during interphase and mitosis. RNAi-mediated-depletion of CENP-

M, CENP-N and CENP-T resulted in the loss of other CenH3CENP-A NAC components (with the

exception of CenH3CENP-A), an enrichment of cells in mitosis, and chromosome missegregation

(Foltz et al., 2006).

Figure m. Figure m. Identification of CenH3/CENP-A-nucleosome

centromere components. Purified LAP-CENP-M, LAP-CENP-N and LAP-

CENP-U(50) complexes revealed the presence of constitutive CENPs

(CENP-I/K/L/O/P/Q) visualized by silver staining. S-tagged components

are indicated as ST. Figure from Foltz et al., 2006.

29

In the same journal issue, another study also reported additional vertebrate kinetochore

proteins (Okada et al., 2006). In this study, the authors performed Co-IPs targeting epitope-

tagged CENP-H and CENP-I followed by mass spectrometry in chicken cells. These analyses

revealed five proteins (CENP-K, CENP-L, CENP-M, CENP-O and CENP-P) interacting with both

CENP-H/I, and which were all constitutively localized to centromeres throughout the cell cycle

(Figure n). Subsequent affinity purification of the human CENP-O and CENP-P homologs from

HeLa cells allowed the identification of two novel kinetochore proteins, CENP-Q and CENP-R,

CENP-N and the previously identified CENP-U (CENP-50) (Minoshima et al., 2005). All of them

also localized constitutively to human centromeres supporting that they are indeed

kinetochore components. Depletion of all identified proteins in both chicken and human cells,

resulted in multiple cell cycle defects to different degrees such as an accumulation of mitotic

cells, cells with hypercondensed chromosomes that failed to congress to the metaphase plate,

or the presence of multipolar spindles. Based on localization studies upon depletion of

individual proteins, the authors established three different protein subgroups: CENP-H (CENP-

H/I/K/L), CENP-O (CENP-O/P/Q/50) and CENP-M classes. Furthermore, upon depletion of

CENP-H/I/K/M in chicken cells the centromeric localization of the microtubule-binding protein

Ndc80Hec1 was reduced. Additionally, CENP-H and CENP-I deficient chicken cells displayed a

lower level of newly synthesized CenH3CENP-A, suggesting that CENP-H/I are required for

directing CenH3CENP-A at centromeres in chicken cells (Okada et al., 2006).

Figure n. Protein purification of CENP-I-GFP and CENP-H-GFP and CENP-H-FLAG in chicken DT40 cells. A) The proteins were

separated on a 4-20% gradient SDS-PAGE gel and visualized by silver staining. The arrows indicate the bands that were excised

A) B)

30

and analyzed by mass spectrometry. B) Mass spectrometry analyses revealed the identity of the excised proteins. Figures

adapted from Okada et al., 2006.

In summary, early genetic screens and large-scale Co-IP experiments combined with mass

spectrometry analyses were critical to the identification of kinetochore components in several

animals and fungi. In the following sections, the role of individual kinetochore components

will be discussed in more detail.

CENP-B

CENP-B together with CENP-A and CENP-C were identified as centromere proteins in the anti-

sera from CREST patients (Earnshaw and Rothfield, 1985). Early studies revealed that CENP-B,

in contrast to CENP-C, was present in both active and inactive centromeres from dicentric

chromosomes, yet with lower signal intensity at the inactive one (Earnshaw et al., 1989).

Thus far, CENP-B is the only known sequence-specific DNA binding protein recognizing a 17

bp DNA motif called CENP-B box (Masumoto et al., 1989). This 17-bp motif is present in

centromeric satellite DNA in all human chromosomes with the exception of the Y chromosome

(Masumoto et al., 1989). CENP-B contains a DNA CENP-B box-binding domain and catalytic

domain of transposases similar to that of the Tigger and pogo transposases (Smit and Riggs,

1996). Based on the high level of sequence identity between CENP-B and the pogo-like

transposases family (Smit and Riggs, 1996), CENP-B is likely domesticated from this family of

transposases and acquired a function at the centromere. CENP-B has been widely described

in mammals (Bejarano and Valdivia, 1996; Bulazel et al., 2006; Casola et al., 2008), but appears

to be absent in other vertebrates (reviewed in Gamba and Fachinetti, 2020) suggesting that

the domestication event occurred in an ancestor to mammals.

The contribution of CENP-B to centromere function has been unclear for a while. CENP-B

knockout (KO) mice are viable (Hudson et al., 1998; Kapoor et al., 1998; Perez-Castro et al.,

1998) and CENP-B was present in both active and inactive centromeres from dicentric

chromosomes, yet with lower signal intensity at the inactive one (Earnshaw et al., 1989).

These observations led to the notion that CENP-B may be dispensable for centromere

function. On the other hand, CENP-B has been proposed to be required for de novo

centromere assembly in human and mammalian artificial chromosomes (HAC/MAC) as CENP-

31

B boxes are sufficient to target CenH3CENP-A assembly (Ohzeki

et al., 2002; Okada et al., 2007). More recently, CENP-B was

also been shown to be involved in CenH3CENP-A and CENP-C

stability through direct protein interactions with CenH3CENP-A

N-terminal tail (Fachinetti et al., 2015; Fujita et al., 2015) and

CENP-C (Suzuki et al., 2004; Fachinetti et al., 2015). Indeed

CENP-B depletion results in reduced centromeric CENP-C

levels without affecting its de novo accumulation at

centromeres (Fachinetti et al., 2015) (Figure o). Together

with the observations that chromosomes without CENP-B

boxes displayed higher missegregation frequencies (and

reduced CENP-C levels), these data supported the notion that

CENP-B is important to ensure faithful chromosome

segregation through the stabilization of other kinetochore

proteins (Fachinetti et al., 2015). Along the same lines, once

the kinetochore is pre-assembled, CenH3CENP-A becomes

dispensable at least for the first following mitotic cycle.

Under these circumstances, it has been shown that CENP-B

becomes essential to stabilize pre-assembled kinetochore

through its interaction with CENP-C to enable chromosome

segregation (Hoffmann et al., 2016). Finally, CENP-B has been

proposed to provide a genetic memory of human centromere

location shown by its ability to occasionally trigger

centromere re-activation and initiate the CenH3CENP-A-based epigenetic loop (Hoffmann et al.,

2020).

CENP-B-like proteins (arisen independently from transposases) have also been identified in

fission yeast (Casola et al., 2008) and Lepidoptera (butterflies and moths) (d’Alençon et al.,

2011). In fission yeast CENP-B homologs: Abp1, Cbh1 and Cbh2 are implicated in centromeric

heterochromatin formation by promoting the recruitment of Swi6 (homolog of

heterochromatin protein1 HP1) (Nakagawa et al., 2002). The function of the lepidopteran

putative CENP-B protein is unknown. Proteomic analyses from our laboratory (unpublished

A)

B)

C)

Figure o. CENP-B is required to ensure

faithful chromosome segregation by

supporting CENP-C maintenance at

centromeres. A) CENP-B depletion results

in increased micronuclei formation. The

yellow arrow indicates a micronuclei in

the CENP-B depleted cell. Scale bar: 5 m.

B) Quantification of the observed:

lagging chromosomes in mitosis and

micronuclei in interphase in cells with or

without CENP-B. Error bars represent the

standard error of the mean of 3

independent experiments. Unpaired t

test: *p < 0.04. C) Quantification of

CenH3CENP-A and CENP-C proteins at

centromeres in cells with or without

CENP-B. Error bars represent the

standard error of the mean. Unpaired t

test: *p < 0.0016. Figures adapted from

Fachinetti et al., 2015.

32

data, personal communication I.A. Drinnenberg) did not reveled evidence for CENP-B at

centromeres indicating that it might have been domesticated for another function.

CENP-C

CENP-C was third antigen recognized by the antisera from CREST patients (Earnshaw and

Rothfield, 1985). Years later, immunoelectron microscopy analyses in HeLa revealed that

CENP-C localized only to active centromeres, most of the times as two discrete dots at the

inner kinetochore plate (Earnshaw et al., 1989). This observation led to the conclusion that

CENP-C is a protein of the inner kinetochore (Saitoh et al., 1992). Subsequent studies revealed

that reduction of CENP-C levels at centromeres by microinjection using anti-CENP-C

antibodies results in cells arrested in mitosis, smaller kinetochores and less microtubules

attached. Taken together these observations suggested that CENP-C plays an essential role in

kinetochore assembly and microtubule-binding (Tomkiel et al., 1994).

CENP-C homologs have been identified in diverse organisms including S. cerevisiae (in this

organism CENP-C is named Mif2) (Brown et al., 1993), chicken cells (Fukagawa, 1997), D.

melanogaster (Heeger, 2005), S. pombe (named Cnp3) (Holland et al., 2005), Xenopus laevis

(frog) (Milks et al., 2009), maize (Dawe et al., 1999) and C. elegans (CENP-C homolog called

HCP-4) (Moore and Roth, 2001). Although, CENP-C’s secondary structure is disordered in most

parts of the protein, there are a couple of highly conserved domains among CENP-C homologs

(reviewed in: Westermann and Schleiffer, 2013; Hara and Fukagawa, 2017). These are: i) a

cupin fold domain located at the C terminus

and necessary for dimerization (Cohen et al.,

2008) and ii) the so-called CENP-C motif that

associates with the C-terminal tail of CenH3

(Carroll et al., 2010; Kato et al., 2013). This

motif is present in all known CENP-C homologs

known including those from budding yeast,

flies (Heeger, 2005), nematodes (Moore and

Roth, 2001) and vertebrates (reviewed in

Talbert et al., 2004). Consistent with the early results shown in HeLa cells (Tomkiel et al.,

1994), disruption of CENP-C in other organisms results in cells arrested in mitosis and

Figure p. Depletion of CENP-C in HeLa cells results in

metaphase arrest. Representative pictures of CENP-C-

depleted cells by anti-CENP-C injection (G panel: DAPI

staining, H: immunostaining for tubulin and I:

immunostaining for centromeres using human ACA serum).

The anti-CENP-C-treated cells remained at metaphase for

more than 2 hours and up to 10 hours, and their

chromosomes failed to align at the metaphase plate and to

segregate to the spindle poles. Scale bar: 10 m. Figure

adapted from Tomkiel et al., 1994

33

chromosome missegregation (Figure p) (Brown et al., 1993; Meluh and Koshland, 1995;

Fukagawa, 1997; Milks et al., 2009).

CenH3 and CENP-C display an interdependent relationship for their recruitment and stability.

While CenH3 nucleosomes serve as scaffold for CENP-C recruitment to centromeres, CENP-C

depletion results in a reduction of existing and nascent pools of CenH3CENP-A in human cells.

This showed that CENP-C is required for CenH3 nucleosome incorporation and stabilization

(Falk et al., 2015; Guo et al., 2017).

In addition to its interaction with CenH3, CENP-C has also been shown to interact with other

CCAN components and is required for their recruitment to kinetochores. For example, CENP-

C has been demonstrated to interact with CENP-L in S. pombe (Tanaka et al., 2009), with CENP-

L/N in chicken and human cells (McKinley et al., 2015; Nagpal et al., 2015) (Figure q-A), and

with CENP-H/I/K/M in human (Figure q-B) and frogs (Klare et al., 2015; Milks et al., 2009). In

more detail, in human cells the PEST domain in the CENP-C N-terminal region interacts with

CENP-H/I/K/M and mutations in this region result in a complete loss of CENP-H/I/K/M and

reduction of CENP-T/W signals at centromeres (Klare et al., 2015). A requirement of CENP-C

for the localization of CENP-T/W in chicken cells may be different in these organisms

(described below on the CENP-T section). Furthermore, data from chicken cells suggests that

CENP-H (Fukagawa, 2001), CENP-K (Kwon et al., 2007) and CENP-I (Nishihashi et al., 2002) are

required for CENP-C centromeric localization in interphase. However during mitosis, CENP-C

can localize to centromeres even in absence of these proteins (reviewed in Cheeseman and

Desai, 2008; Nagpal et al., 2015). Similar observations have been reported in human cells

where mitotic localization of CENP-C is independent of other CCAN proteins, while in

interphase CENP-C requires CENP-N and CENP-H/I/K/M for its localization (McKinley et al.,

2015) (Figure q-C). Thus, the interactions between the kinetochore subcomplexes seems to

be rather complex and dynamic during the cell cycle instead of a linear hierarchy.

CENP-C also bridges the inner to the outer kinetochore. The N-terminal tail of CENP-C interacts

with the outer kinetochore Mis12 complex (Mis12C) in human (Screpanti et al., 2011) and D.

melanogaster cells (Przewloka et al., 2011). Indeed CENP-C depletion results in reduction of

Mis12C proteins (Liu et al., 2006; Kwon et al., 2007) and Ndc80Hec1 signals (Okada et al., 2006).

Similar results have been observed in X. laevis where Mis12C components (Dsn1 and Nnf1)

34

require CENP-C for their targeting to centromeres (Milks et al., 2009). Likewise, in human cells

CENP-C KO results in reduction of Knl1, Dsn1 and Ndc80Hec1 levels (McKinley et al., 2015).

Figure q. CENP-C interacts with other CCAN components. A) Middle portion of CENP-C interacts with CENP-L/N. Histidine pull

down in chicken cells of CENP-C middle portion with CENP-Nct/L complex. CENP-C middle portion (amino acids: 166-324, CENP-

C166-324) was fused to maltose binding protein (MBP) and histidine (His) tags. MBP-CENP-C166-324-His was immobilized on Ni

(nickel)-Sepharose beads and used as bait to pull-down MBP-CENP-Nct/L. Figure from adapted Nagpal et al., 2015. B) Size-

exclusion chromatography (SEC- to separate proteins based on size and shape) elution profile from human cells. CENP-C2–545

and CENP-H/I/K/M form a stable complex. Figure adapted from Klare et al., 2015. C) Schematic of the interactions of CENP-C

with CENP-L/N and CENP-H/I/K/M subcomplexes in mitosis and interphase in human cells. Figure from McKinley et al., 2015

Altogether, these findings established CENP-C as a major player for kinetochore assembly

directly bridging CenH3 containing nucleosomes from the inner to the outer kinetochore.

CENP-T/W/S/X

CENP-T was identified together with CENP-M, CENP-N and CENP-U as centromere

components of the CenH3CENP-A proximal nucleosome associated complex (CENP-A NAC) (Foltz

et al., 2006). A parallel study also identified CENP-T (named ICEN22) as CenH3CENP-A chromatin

associated protein. This study also reported that RNAi-mediated depletion of CENP-T results

in chromosome congression errors, reminiscent to phenotypes observed upon depletion of

other centromeric components (Izuta et al., 2006). In addition, both studies reported the

constitutive presence of CENP-T at centromeres throughout the cell cycle (Foltz et al., 2006;

Izuta et al., 2006).

A) C)B)

35

Years later, CENP-W was identified

as direct binding partner of CENP-T

(Foltz et al., 2006; Hori et al.,

2008a). In fact, both proteins

contain histone fold domains that

form a tight tetrameric complex

that associates with DNA thereby

contributing to the attachment of

the kinetochore complex to

centromeres (Hori et al., 2008a;

Nishino et al., 2012). CENP-T and

CENP-W depletions in chicken and

HeLa cells result in cells arrested in

mitosis, chromosome failure to

congress and hypercondensed

chromosomes indicating that these proteins are essential for chromosome alignment and

mitotic progression (Hori et al., 2008a) (Figure r). A CENP-T/CENP-W dimer has also been

shown to interact with two additional histone fold proteins, CENP-S and CENP-X to form a

tetrameric complex (Amano et al., 2009). Nevertheless, the extent to which a CENP-T/W/S/X

complex forms at centromeres is controversial due to the fact that CENP-S and CENP-X

deletions are rather mild compared to CENP-T and CENP-W deletions (Amano et al., 2009). In

addition, CENP-S and CENP-X has also been shown to interact with Fanconi anemia

complementation group M (FANCM) protein complex and implicated in DNA damage

response and repair (Singh et al., 2010; Yan et al., 2010).

The CENP-T/CENP-W heterodimer interacts with multiple components at the inner

kinetochore. For example, CENP-T/CENP-W directly interact with CENP-H/I/K/(M) complex in

budding yeast and human cells (Basilico et al., 2014; Pekgöz Altunkaya et al., 2016). Both

complexes are mutually exclusive for their recruitment (Basilico et al., 2014; McKinley et al.,

2015; Pekgöz Altunkaya et al., 2016). Moreover, it was found that the direct interaction

between CENP-T/W and the CENP-H/I/K/M complex (Basilico et al., 2014; Klare et al., 2015)

has a lower affinity than the binding between CENP-C and CENP-H/I/K/M complex (Klare et

Figure r. CENP-T/W dimer is important for chromosome segregation.

Depletion of CENP-T and CENP-W results in misaligned chromosomes and

cell-arrest in mitosis. Top) Quantification of phenotypes observed in

mitosis and interphase upon depletion of CENP-T and CENP-W in chicken

DT40 cells. Bottom) Representative pictures of control cells (-tet), and

CENP-W or CENP-T-depleted cells (+tet) (DAPI in blue and anti-tubulin in

green). Scale bar 10m. Figures adapted from Hori et al., 2008a

36

al., 2015). As mentioned in the previous section on CENP-C, in human cells CENP-C depletion

results in the mislocalization of CENP-H/K and CENP-T/W, therefore both the CENP-H/I/K/M

and CENP-T/W complexes appear to localize downstream of CENP-C (Carroll et al., 2010;

Basilico et al., 2014; Klare et al., 2015; McKinley et al., 2015). The CENP-T connectivity with

other CCAN components might be slightly different in chicken cells where it has been shown

that upon depletion of CENP-C, levels of CENP-T/W at centromeres remain unaltered (Hori et

al., 2008a). Likewise in CENP-W-deleted cells, CENP-C localization at centromeres remains

unaffected. Moreover, it was shown in chicken cells that CENP-K, CENP-S and CENP-U proteins

are no longer at centromeres upon deletion of CENP-W (Hori et al., 2008a). In contrast, in

CENP-H-depleted cells CENP-T and CENP-W levels while reduced, remain present at the

centromeres (Hori et al., 2008a). This observation suggests that CENP-T/W localization

depends at least partially on the CENP-H/I/K/(M) complex. While direct protein interactions

have not been demonstrated, CENP-T localization appears to require the CENP-L/N (McKinley

et al., 2015; Samejima et al., 2015) in human cells and the N-terminal tail of CenH3 in fission

yeast (Folco et al., 2015).

In addition to CENP-C, CENP-T is also a key player for the recruitment of the outer kinetochore.

In fact, artificial tethering of the N-terminus of CENP-T or CENP-C are sufficient, in the absence

of CenH3, to recruit outer kinetochore components capable to attach to spindle microtubules

(Gascoigne et al., 2011) (Figure s-A). While CENP-C recruits the Mis12C (Przewloka et al., 2011;

Screpanti et al., 2011; Richter et al., 2016), the extended N-terminal region of CENP-T is

sufficient to recruit the microtubule-binding protein Ndc80Hec1 in a CDK-dependent

phosphorylation manner (Gascoigne et al., 2011). In fact, CENP-T N-terminal binds directly the

Spc24/Spc25 complex of the Ndc80 in human, chicken and budding yeast (Bock et al., 2012;

Schleiffer et al., 2012; Nishino et al., 2013). In addition, CENP-T also recruits the Mis12

complex through direct protein interaction or indirectly through the Ndc80 complex (Huis in

’t Veld et al., 2016; Rago et al., 2015) (Figure s-B). A recent study in chicken cells (Hara et al.,

2018) showed that CENP-T binds to two copies of the Ndc80C, instead of the three Ndc80C

copies that human CENP-T binds (Huis in ’t Veld et al., 2016). In DT40 cells, one Ndc80C copy

binds directly to CENP-T N-terminal tail and the second copy binds indirectly through the

Mis12C. Both interactions are essential as cells expressing either CENP-T truncated at the N-

terminus or Dsn1 mutant unable to bind to the Ndc80C died. Moreover, Hara et al., showed

37

that in chicken cells the Ndc80C preferentially binds to CENP-T over CENP-C during mitosis.

With regards of the Mis12C, the authors found that Mis12C is mainly associated with CENP-C

during late G2/prophase, then during mitosis Mis12C interacts with both CENP-C and CENP-T.

In fact, during prometaphase to anaphase the binding levels between Mis12C and CENP-C

decrease, while Mis12C binding levels with CENP-T remain constant. Furthermore, the

Mis12C-CENP-C interaction is dispensable for cell viability and mitotic progression (Hara et al.,

2018). Taken together CENP-C and CENP-T constitute two independent pathways to recruit

the microtubule-binding outer kinetochore at mitosis.

Figure s. CENP-T acts as outer kinetochore recruiter. A) Representative immunofluorescence images showing the localization

of i) artificially tethered GFP-LacI (top panel) or GFP-CENP-TDC-LacI (bottom panel) to LacO arrays, and ii) outer kinetochore

proteins: Dsn1 (member of the Mis12 complex), KNL1 and Ndc80Hec1 (member of the Ndc80 complex). On the right)

Quantification of the ratio of: the fluorescence of the indicated kinetochore proteins (Dsn1, KNL1 and Ndc80Hec1) at

endogenous kinetochores/ versus the fluorescence of these kinetochore proteins at ectopic foci in cells expressing LacI fusion

proteins. Figures adapted from Gascoigne et al., 2011. B) Representative immunofluorescence images showing the localization

pattern of GFP-CENP-T-LacI and outer kinetochore proteins. Numbers in white indicate number of mitotic cells showing

colocalization. Scale bar: 5m. Figure adapted from Rago et al., 2015.

CENP-H/I/K/M

CENP-H was identified in mouse cells as a kinetochore protein with a coiled-coil structure that

localizes constitutively to centromeres throughout the cell cycle (Sugata et al., 1999).

Subsequent immunofluorescence assays revealed that human CENP-H localizes at the inner

kinetochore at active centromeres and interacts with both CENP-C and CenH3CENP-A (Sugata et

al., 2000). These observations suggested that CENP-H could be essential for kinetochore

assembly. Indeed CENP-H depletion in chicken and human cells results in metaphase arrest

and chromosome misalignment followed by an increase in apoptotic cells (Fukagawa, 2001;

Foltz et al., 2006) (Figure t). Furthermore, CENP-H is necessary for CENP-C recruitment but

dispensable for CenH3 centromeric localization (Fukagawa, 2001; Foltz et al., 2006). Loss of

A) B)

38

chicken CENP-H also caused a reduction of Ndc80Hec1 levels

at centromeres to similar levels as observed upon CENP-C

depletion (Okada et al., 2006). Thus, CENP-H is required for

proper chromosome segregation and outer kinetochore

assembly.

CENP-I was first described in fission yeast (named Mis6 in

this organism) as an essential kinetochore protein required

for cell vialibily. It has been reported that CENP-IMis6

depletion results in chromosome misegregation and

reduced sporulation in fission yeast (Saitoh et al., 1997).

Years later, the chicken CENP-I homolog was identified

based on sequence similarity and characterized as a

kinetochore protein that localizes to centromeres

throughout the cell cycle. Similar to CENP-I

depletion in fission yeast, CENP-I is also essential

in chicken cells and its loss causes mitotic arrest

and chromosome misalignment (Figure u).

Additionally, upon CENP-I depletion in chicken

cells, CENP-C staining was diffused while CenH3

signals were unaffected in interphase. Thus, like

CENP-H, vertebrate CENP-I is also required for

CENP-C recruitement (Nishihashi et al., 2002). In

addition, CENP-I and CENP-H localization has

been shown to be interdependent in chicken

and human cells as depletion of one results in

the loss of the other (Nishihashi et al., 2002;

Foltz et al., 2006; Izuta et al., 2006). Later studies

performing proteomic analyses further

confirmed CENP-I’s role at the centromere

Figure t. CENP-H deletion results in

chromosome missegregation.

Representative images of wild-type (-tet,

top row) and CENP-H-deleted (+tet)

chicken cells stained with anti-tubulin

(green) and DAPI (blue). In treated cells,

chromosomes failed to align at the

metaphase plate (middle row, misaligned

chromosomes are indicated by the white

arrows). Furthermore, CENP-H-deleted

cells displayed monopolar and multipolar

spindles (bottom row). Scale bar: 10 m. Figure from Fukagawa et al., 2001.

Figure u. CENP-I-deficient cells display

misaligned chromosome leading to chromosome

missegregation. Representative images of wild-

type (top row) and CENP-I-deficient (2nd, 3rd and

4th rows) chicken cells stained with anti-tubulin

(green) and DAPI (blue). In treated cells,

chromosomes failed to aligned at the metaphase

plate (2nd and 3rd rows, misaligned or lagging

chromosomes are indicated by the white

arrows). Moreover, CENP-I-deleted cells displayed

monopolar and multipolar spindles (bottom row).

Scale bar: 10 m. Figure from Nishihashi et al., 2002.

39

(Obuse et al., 2004) and recovered it as part

of the CenH3CENP-A nucleosome associated

complex in HeLa cells (Foltz et al., 2006).

Mass spectrometric analysis of CENP-H and

CENP-I IPs in chicken and human cells (Okada

et al., 2006), tandem affinity purification of

CenH3CENP-A nucleosomes (Foltz et al., 2006)

and ChIP with anti-CENP-A antibody (Izuta et

al., 2006) allowed the identification of their

interacting proteins. Among those, CENP-M

and CENP-K were identifed, the latter one

with low sequence similarity to fission yeast

Sim4 homolog (Okada et al., 2006). As CENP-

H/I, both CENP-K and CENP-M localize to

centromeres throughout the cell cycle and

are essential for cell viability. Loss of CENP-K

resulted in similar phenotypes observed

upon CENP-H and CENP-I depletions,

including an accumulation of mitotic cells,

hypercondensed chromosomes,

chromosome misaligments and cells with multipolar spindles (Figure v-A) (Foltz et al., 2006;

Izuta et al., 2006; Okada et al., 2006). CENP-M loss also caused similar defects (Figure v-B)

(Foltz et al., 2006; Izuta et al., 2006; Okada et al., 2006; Basilico et al., 2014). The accumulation

of mitotic cells upon CENP-K depletion however was milder and at later time points (Okada et

al., 2006). These first observations led to the notion that CENP-K was part of CENP-H/I

complex, but CENP-M represented another class of kinetochore proteins (Okada et al., 2006).

However depletion of each component (CENP-H/I/K/M) results in the loss of other complex

components showing that their localization is interdependent (Foltz et al., 2006; Izuta et al.,

2006; Okada et al., 2006).

Figure v. CENP-M and CENP-K depletions led to chromosome

missegregation. A) Representative images of control (top row)

and CENP-M-depleted (bottom row) HeLa cells stained with

anti-tubulin (green), DAPI (blue) and anti-centromere

autoimmune serum (ACA) to mark centromeres (red). CENP-M

siRNA-treated cells displayed misaligned chromosomes and

multipolar spindles. Scale bar: 5 m. Figure adapted from Foltz

et al., 2006. B) Representative images of control (top row) and

CENP-K-deficient (bottom row) chicken DT40 cells stained with

anti-tubulin (green) and DAPI (red). The anti-centromere

autoimmune serum (ACA) staining is shown on the left

column. CENP-K RNAi-treated cells displayed misaligned

chromosomes. Figure adapted from Okada et al., 2006.

A)

B)

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40

The CENP-H/I/K/M subcomplex is necessary for recruitment of other CCAN components. In

budding yeast, CENP-ICtf3 is required for CENP-TCnn1 centromere localization, which in turn

connects to the amino-termini of CENP-HMcm16 and CENP-KMcm22 (Pekgöz Altunkaya et al.,

2016). In contrast to earlier observations using CENP-I along (Nishihashi et al., 2002), CENP-

H/I/K/M mutants displayed lower levels of newly synthetized CenH3 at centromeres in

vertebrate cells suggesting that CENP-H/I/K/M is required for CenH3 centromere targeting

(Okada et al., 2006). Similar observations were also reported in fission yeast upon CENP-IMis6

depletion (Takahashi et al., 2000). Furthermore, CENP-H/I/K/M is required for CENP-T/W

(Basilico et al., 2014), CENP-L/N (McClelland et al., 2007; Carroll et al., 2009) and CENP-C

(Fukagawa, 2001) recruitment to kinetochores. The localization dependency of other CCAN

components on CENP-H/I/K/M can be different in different cell cycle stages. While CENP-

H/I/K/M is required for CENP-C assembly during interphase in chicken cells, in mitosis, CENP-

C localizes to centromeres even in absence of CENP-H/I/K/M (Kwon et al., 2007; Cheeseman

et al., 2008) (Figure q-C).

In turn, the recruitment of CENP-H/I/K/M requires other CCAN proteins including CENP-T

(Izuta et al., 2006; Hori et al., 2008a; Gascoigne et al., 2011), CENP-N (Izuta et al., 2006; Carroll

et al., 2009), CENP-L (Izuta et al., 2006) and CENP-C (Klare et al., 2015). In human cells, CENP-

H/I/K/M interacts with the PEST domain of CENP-C, which is

necessary to recruit CENP-H/I/K/M to centromeres (Klare et

al., 2015). Similarly, CENP-H/I/K/M recuitment also depends

on CENP-C in X. laevis (Milks et al., 2009; Carroll et al., 2010),

while upon deletion of CENP-C in chicken cells reduced

CENP-H/I/K/M signals continue to be at centromeres

(Fukagawa, 2001; Kwon et al., 2007). In addition to its role

in CCAN assembly, CENP-H/I/K/M complex also interacts

with the outer kinetochore. In particular, CENP-H (Mikami et

al., 2005; Pekgöz Altunkaya et al., 2016) and CENP-K

(Cheeseman et al., 2008) have been shown to interact with

Ndc80Hec1 protein from the Ndc80 complex (Ndc80C).

Moreover, reduced levels of Ndc80 upon CENP-H/-K (Figure

RNAi

Figure w. CENP-K is direct the

kinetochore localization of Ndc80.

Representative immunofluorescence

images of control (top row) and CENP-K-

depleted (bottom row) HeLa cells

stained with anti-Hec1 (human Ndc80-

hNdc80). The white numbers in the

bottom right indicate the kinetochore

fluorescence intensities and the

standard error (expressed as

percentage) relative to control cells.

Figure adapted from Cheeseman et al.,

2008.

41

w) depletion led to the conclusion that CENP-H/I/K/M is involved in the recruitment of the

Ndc80C.

Biochemical analyses of the CENP-H/I/K/(M) complex have revealed insights into its structure

but has been challenged due to technical problems purifying the complex. For example, in

human cells CENP-I purification requires co-expression with the subcomplex proteins CENP-H

and CENP-K. The stability of this tetrameric complex further requires co-expression with CENP-

M (Basilico et al., 2014). Purification and crystal structure of the CENP-H/I/K/M subcomplex

revealed that CENP-H/K heterodimer interacts directly with the CENP-I N-terminal region, but

not with CENP-M (Basilico et al., 2014). Instead, CENP-I connects CENP-M with CENP-H/K.

These biochemical analyses further revealed that CENP-M is structurally related to small

GTPases, however it has lost sequence motifs required for GTP binding and hydrolysis by small

G-proteins (Basilico et al., 2014). Interestingly, CENP-M has no known homolog in yeast

(reviewed in Westermann and Schleiffer, 2013) raising the possibility that in yeast CENP-H/I/K

is indeed a tetramer. The recent crystal structure of CENP-I N-terminal tail from Chaetomium

thermophilum and CENP-H/K heterodimer from Thielavia terrestris revealed that the carboxyl-

terminal of CENP-H contains two alpha-helixes that interact with both carboxyl-terminal of

CENP-K and the amino-terminal of CENP-I, resulting in a stable complex where CENP-H is

located between CENP-K and CENP-I (Hu et al., 2019). Nevertheless, whether this

configuration is conserved in other organisms including vertebrates is still unknown.

CENP-L/N

CENP-L and CENP-N form the CENP-L/N subcomplex (Carroll et al., 2009; Weir et al., 2016)

that localize to centromeres throughout the cell cycle similar to other CCAN components.

These kinetochore components were first identified as CenH3CENP-A nucleosome interacting

proteins in human cells (Foltz et al., 2006; Izuta et al., 2006) and CENP-H/I/K/M interacting

proteins in chicken cells (Okada et al., 2006).

Depletions of CENP-L or CENP-N result in congression defects, mitotic progression delay,

lagging chromosomes and failure in spindle assembly (Foltz et al., 2006; Izuta et al., 2006;

Okada et al., 2006; McClelland et al., 2007; McKinley et al., 2015) (Figure x). Hence CENP-L/N

are required for proper chromosome alignment and chromosome segregation.

42

Figure x. CENP-N and CENP-L are required for accurate chromosome segregation. A) Representative immunofluorescence

images of control (top row) and CENP-N-deficient (2nd and 3rd rows) HeLa cells. CENP-N knockout results in an increase of

misaligned chromosomes at the metaphase plate and multipolar spindles. Right side) Quantification of the percentage of cells

displaying these mitotic defects Scale bar: 5 m. Figure adapted from McKinley et al., 2015. B) Representative

immunofluorescence images of control (top row) and CENP-L-deficient (bottom row) DT40 cells. As CENP-N KO, CENP-L

depletion results in misaligned chromosomes at the metaphase plate. Anti-tubulin staining is in green and DAPI in red. The

anti-centromere autoimmune serum (ACA) staining is shown on the left column. Figure adapted from Okada et al., 2006.

CENP-L/N are also important for CCAN assembly in a mutually-exclusive manner. Indeed

CENP-L/N loss abolishes CENP-H/I/K/M, CENP-T/W, CENP-O, CENP-P and CENP-Q localization

(Carroll et al., 2009; McKinley et al., 2015; Weir et al., 2016) while in turn these proteins are

also required for CENP-L/N localization at kinetochores (Carroll et al., 2009). The interaction

of CENP-L/N to these CCAN components is mediated through CENP-L (Carroll et al., 2009).

CENP-N in contrast directly interacts with CenH3-associated chromatin through its C-terminal

region. While CENP-C interacts with the C-terminal tail of CenH3, CENP-N binds to the CenH3

CATD domain (Pentakota et al., 2017). Moreover, it has been proposed that the specificity of

CENP-N-CenH3 interaction is mainly driven by the structural conformation of the CATD of

CenH3CENP-A rather than by sequence (Carroll et al., 2009; McKinley et al., 2015).

A recent study based on the crystal structure of the CENP-N:CenH3CENP-A nucleosome, further

reveal that both CenH3CENP-A and CENP-C are required for CENP-N recruitment to kinetochores

(Carroll et al., 2009). In turn, CENP-C (Nagpal et al., 2015; McKinley et al., 2015) and newly

synthetized CenH3CENP-A levels (Foltz et al., 2006; Izuta et al., 2006) at centromeres are reduced

upon CENP-N loss. Later studies further distinguished between different cell cycle stages and

found that, similar to the effect upon CENP-I depletion, CENP-N depletion reduces CENP-C

localization to centromeres during interphase but not during mitosis. This observation led to

A) B)

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43

the notion that CENP-N (as CENP-I, discussed above) stabilizes CENP-C during interphase in

human and chicken cells (Minoshima et al., 2005; Okada et al., 2006) but is not required for

CENP-C’s stability during mitosis (Figure q-C).

CENP-O/P/Q/U/R

As CENP-L/N, the CENP-O/P/Q/U/R complex was found to co-purify with other CenH3CENP-A

nucleosomes interacting proteins in human cells (Minoshima et al., 2005; Foltz et al., 2006;

Izuta et al., 2006; Okada et al., 2006) and as interacting proteins with the CENP-H/I/K/M

complex in chicken cells (Okada et al., 2006; Hori et al., 2008b; McKinley et al., 2015; Pesenti

et al., 2018). As other CCAN components, these proteins are also constitutively present at

centromeres during the cell cycle (Minoshima et al., 2005; Foltz et al., 2006; Izuta et al., 2006;

Okada et al., 2006). The localization of components of the CENP-O/P/Q/U/R complex is

interdependent on one another, with the exception of CENP-R in chicken cells that does not

appear to be necessary for the localization of the respective other components (Okada et al.,

2006; Hori et al., 2008b; McKinley et al., 2015; Pesenti et al., 2018). This complex counterpart

in S. cerevisiae is the COMA complex composed by CENP-PCtf19, CENP-QOkp1, CENP-OMcm21 and

CENP-UAme1, while CENP-R has no known homolog in yeast (De Wulf, 2003).

Depletion of CENP-U (previously named CENP-50 (Minoshima et al., 2005)) in human cells

results in lagging chromosomes, congression defects and unstable microtubule attachment

(Foltz et al., 2006) (Figure y-A). Likewise, CENP-O/P/Q/U loss in chicken cells also results in

chromosomes that fail to congress at the metaphase plate (Minoshima et al., 2005; Okada et

al., 2006; Hori et al., 2008b). Thus, the CENP-O/P/Q/U/R proteins are required for a proper

chromosome alignment (Figure y-B). Nevertheless, CENP-O/P/Q/U/R loss in chicken cells

causes mild mitotic defects compared to those observed upon depletion of other CCAN

components and does not affect viability. Taken together, these observations led to the notion

that in vertebrates CENP-O/P/Q/U/R subcomplex is not essential, but instead could fine-tune

proper chromosome segregation (Hori et al., 2008b). While CENP-O/P/Q/U/R KO cells are

viable, CENP-U KO mice die during early embryogenesis. Thus, in contrast to the in vitro

results, these proteins appear to be essential in vivo at least in certain cell types or

developmental stages (Hori et al., 2008b).

44

Figure y. CENP-O/P/Q/U/R subcomplex as other CCAN components is required for accurate chromosome segregation. A) Top:

Live cell time-lapse on control and CENP-U-depleted HeLa cells (2nd and 3rd rows). CENP-U-deficient cells displayed mitotic

defects as: misaligned and lagging chromosomes, and arrest in mitosis. Scale bar: 5 m. Bottom: quantification of the mitotic

errors observed upon CENP-U depletion. Figures adapted from Foltz et al., 2006. B) CENP-Q KO in DT40 cells result in mitotic

defects. Top row) Representative immunofluorescence images of CENP-Q-deficient cells with misaligned chromosomes at the

metaphase plate (white arrows) and multipolar spindles (white arrowheads). DAPI staining is in blue and anti-tubulin in green.

Middle and bottom rows) Live time-lapse of control and CENP-Q-KO DT40 cells. CENP-Q-deficient cells displayed misaligned

chromosomes and took longer to complete mitosis compared to control cells. Bottom) Quantification of the time progression

in mitosis in control and CENP-Q KO cells analyzed by live cell time-lapse. Figures adapted from: Hori et al., 2008b.

CENP-O/P/Q/U/R subcomplex requires CENP-H/I/K/M, CENP-L/N and CENP-C for its

centromeric recruitment in vertebrate cell lines (Minoshima et al., 2005; Foltz et al., 2006;

Okada et al., 2006; Hori et al., 2008b; Pesenti et al., 2018). Indeed, a recent study revealed

that CENP-O/P dimer directly interacts with a composite interphase created by CENP-H/I/K/M

and CENP-L/N complexes. Then, only in presence of both CENP-H/I/K/M and CENP-L/N, CENP-

O/P dimer binds to CENP-C. Hence, CENP-O/P dimer was proposed to work as a bridge

between CENP-C/CENP-H/I/K/M and the rest of the CENP-O/P/Q/U/R subcomplex (Pesenti et

al., 2018). In contrast, reduction of CENP-U does not impact the centromeric localization of

neither CenH3CENP-A, CENP-B, Ndc80Hec1,Mis12, CENP-H/I/K/M nor CENP-N (Minoshima et al.,

2005; Foltz et al., 2006; Okada et al., 2006). Similarly in CENP-O depleted cells, centromere

levels of CenH3CENP-A, CENP-C,, CENP-H/I/K/M, CENP-L/N and Ndc80Hec1 remain unaffected

(Hori et al., 2008b; McKinley et al., 2015). Taken together, these observations led to the

conclusion that the CENP-O/P/Q/U/R subcomplex is the most downstream subcomplex of the

A) B)

45

CCAN hierarchy in vertebrates. In yeast, however loss of CENP-PCft19 or CENP-OMcm21 results in

a reduction of CENP-LIml3/CENP-NChl4, CENP-H/I/KCtf3 and CENP-TCnn1/CENP-WWip1 complexes

(Pekgöz Altunkaya et al., 2016). CENP-O/P/Q/U thus appears to be essential in budding yeast

in contrast to vertebrate cells.

CENP-O/P/Q/U/R complex has microtubule-binding activity (Amaro et al., 2010; Bancroft et

al., 2015; Hua et al., 2011; Pesenti et al., 2018). However, this is limited to CENP-Q/U dimer,

in particular to the highly basic and disordered N-terminal region of CENP-Q that appears to

be necessary for this activity (Pesenti et al., 2018). Interestingly, CENP-Q N-terminal region is

quite similar to that of the microtubule-binding protein Ndc80. In fact, the N-terminal region

of Ndc80 was shown to be sufficient to rescue the microtubule-binding activity when it is fused

to CENP-Q Nter (Pesenti et al., 2018).

In yeast, the functional relevance of the CENP-O/P/Q/UCOMA complex appears to be different

compared to vertebrates. For instance, CENP-UAme1 contains a Mis12Mtw1-binding motif that is

both sufficient and necessary for this interaction. Indeed, yeast strains expressing CENP-UAme1

mutant protein without the Mis12Mtw1 receptor motif failed to make viable haploid spores,

thus this binding motif in CENP-UAme1 is essential in yeast (Hornung et al., 2014). Several

studies have focused on elucidating the structure and interaction of the CENP-O/P/Q/UCOMA

complex within its units and its interaction with other CCAN subcomplexes (Hornung et al.,

2014; Pekgöz Altunkaya et al., 2016; Schmitzberger et al., 2017; Hinshaw and Harrison, 2019;

Fischböck-Halwachs et al., 2019). It has been reported that CENP-UAme1 and CENP-QOkp1 can

bind to DNA and interact with CENP-CMif2 and CenH3Chl4 (Hornung et al., 2014; Schmitzberger

et al., 2017; Hinshaw and Harrison, 2019). Furthermore, CENP-UAme1 and CENP-QOkp1, together

with the yeast-specific Nkp1/2 subunits, directly interact with the Mis12MIND complex through

their N-terminal extensions (Dimitrova et al., 2016; Hinshaw and Harrison, 2019). The other

two COMA subunits, CENP-PCft19 and CENP-OMcm21 contain RWD domains which act as

interaction surface with CENP-UAme1 and CENP-QOkp1 (Schmitzberger and Harrison, 2012).

Furthermore, CENP-PCft19 and CENP-OMcm21 recruit the CENP-H/I/KCtf3 complex through the

Mcm21CENP-O N-terminal extension (Hinshaw and Harrison, 2019). Taken together, these

observations show that homologous kinetochore subcomplexes can have different functions

and interactions in different organisms.

46

47

Outer kinetochore

While the CCAN is constitutively located at centromeres and

constitutes the DNA-proximal region of the kinetochore, the

outer kinetochore is only recruited to centromeres upon entry

to mitosis. For their recruitment outer kinetochore

components are in direct contact with multiple inner

kinetochore proteins. Once assembled, numerous outer

kinetochore components directly bind microtubules from the

mitotic spindle in a dynamic manner and serve as platform for

the recruitment of components of the spindle assembly

checkpoint, thus contributing to kinetochore bi-orientation and

faithful segregation of the chromosomes during cell division

(reviewed in Cheeseman, 2014).

The outer kinetochore is composed of a conserved 10-group

proteins which are organized in subcomplexes: the Knl1, Mis12

and Ndc80 complexes, known together as the KMN network

(DeLuca and Musacchio, 2012) (Figure z). Among these

subcomplexes, the Ndc80 complex acts as the core player of the

microtubule attachment activity (Cheeseman et al., 2006). This

subcomplex is composed by one copy out of four subunits:

Ndc80 (called Hec1 in humans), Spc24, Spc25 and Nuf2 proteins

(reviewed in Cheeseman, 2014). Depletion of Ndc80C subunits

results in loss of the other Ndc80C components, mitotic arrest,

multi-spindle defects, chromosome congression failure, misaligned chromosomes (Janke,

2001; Wigge and Kilmartin, 2001; McCleland, 2003; Bharadwaj et al., 2004; reviewed in Kline-

Smith et al., 2005) (Figure aa) and loss of checkpoint protein recruitment including Mad1 and

Mad2 (Ciferri et al., 2005). Taken together these observations showed that the Ndc80C is

required for chromosome congression and cell cycle checkpoint control.

Figure z. Schematic model of the outer

kinetochore. the CENP-T/W dimer

(WT) and CENP-C (C) make contact

with the outer kinetochore. As

discussed above, the C-terminal

region of CENP-T associates with H3-

containing nucleosomes (H3) and by

its N-terminal region, CENP-T makes

contacts with the Ndc80 complex

(Ndc80-C) and the Mis12 complex

(Mis12C). In contrast, CENP-C

associates with CenH3CENP-A-

containing nucleosomes (CA), and the

CENP-C N-terminal region makes

contacts with the Mis12C. The Knl1

complex (KNL1-C, composed by Knl1

and Zwint-1) in turn, directly interacts

with the Mis12C (via Knl1) and has

microtubule-binding activity. Spindle

microtubules are represented in light

and dark green lines. Figure adapted

from DeLuca and Musacchio, 2012.

48

Ndc80C has an elongated 50 nm long rod-shaped

structure with globular regions at each end and a

coiled-coil core (Wei et al., 2005). In the complex,

the globular N-terminal tail of Spc24/Spc25 dimer

interacts with the globular C-terminal region of

Ndc80/Nuf2 dimer (Ciferri et al., 2005). While the

Spc24/25 dimer interacts with CENP-T (Nishino et

al., 2013), Ndc80/Nuf2 dimer is in direct contact

with spindle microtubules (Wei et al., 2006).

Biochemical reconstitution analyses and

structural analyses of the Ndc80C showed that

this architecture is conserved in yeast and

humans (Ciferri et al., 2005; Wei et al., 2005,

2006).

The unstructured N-terminal tails and calponin

homology (CH) domains (Ciferri et al., 2008;

Alushin et al., 2010) (found in other actin and

microtubule-binding proteins (reviewed in

Gimona et al., 2002)) of Ndc80Hec1 and Nuf2

regulate microtubule plus-end attachment

stability and plus-end assembly dynamics in an Aurora B kinase phosphorylation-dependent

manner (Cheeseman et al., 2006; DeLuca et al., 2006; Wei et al., 2007; Guimaraes et al., 2008;

Miller et al., 2008). At early mitosis when microtubule-kinetochore attachments errors are

frequent, Ndc80 N-terminal tail is highly phosphorylated by Aurora B. This results in a

reduction of positive charges and weakened microtubule attachments to enable error

correction (DeLuca et al., 2011). In contrast, once chromosomes are bioriented,

dephosphorylated Ndc80 contributes to increase microtubule-kinetochore attachments

stability (Cheeseman et al., 2006; DeLuca et al., 2011; Umbreit et al., 2012) and progression

into anaphase.

A)

B)

Figure aa. Ndc80 complex is required for accurate

chromosome segregation and correct spindle

morphology. A) Representative immunofluorescence

pictures of control (left column) HeLa and HeLa Spc25-

depleted cells (2nd-4th columns). Anti-tubulin in red (1st

row) and DAPI in blue (2nd row). Depletion of Spc25

results in multipolar spindles (2nd column),

chromosome alignment defects (3rd column) and

elongated spindles (4th column). Figure from

Bharadwaj et al., 2004. B) Representative pictures of

Xenopus XTC Ndc80-deficient (top) and control cells

(bottom). Mitotic Xenopus XTC cells injected with

function-blocking anti-Ndc80 antibodies failed to align

chromosomes at the metaphase plate and showed

changes in spindle structure compared to control cells.

Scale bar: 10 m. Figure from McCleland et al., 2003

49

While the Ndc80C provides the main microtubule-attachment site at the outer kinetochore,

another KMN network component, Knl1 (of the KNL1 complex that also contains Zwint-1

(reviewed in Nagpal and Fukagawa, 2016)) also binds microtubules independently of Ndc80C

(Cheeseman et al., 2006). RNAi-based depletion of Knl1 in C. elegans results in chromosome

segregation defects, premature spindle elongation and loss of kinetochore function-

phenotypes that have been classified as so-called “kinetochore-null” effects resembling those

upon CenH3 and CENP-C depletion in C. elegans (Desai, 2003; Oegema et al., 2001). Based on

this kinetochore null phenotype, Knl1 was named (Desai, 2003). Similar to other outer

kinetochore proteins, Knl1 loss does not affect centromeric targeting of CCAN proteins (Desai,

2003). In contrast, Ndc80C levels at kinetochores are reduced after Knl1 depletion

(Cheeseman et al., 2008). Thus, Knl1 is required for outer kinetochore assembly.

Knl1 is the largest core kinetochore protein with a 22 nm elongated structure that is

intrinsically disordered in large parts. The C-terminal tail is more defined and consists of a

coiled coil domain (Petrovic et al., 2010) and tandem RWD domains (Petrovic et al., 2014)

resembling those found in CENP-O, CENP-P (Schmitzberger and Harrison, 2012) and Spc24/25

(Nishino et al., 2013). Through its coiled coil domain Knl1 interacts with its Knl1 complex

binding partner Zwint-1 that is involved in spindle assembly checkpoint and kinetochore-

microtubule attachment (Petrovic et al., 2010). Through its RWD domains Knl1 interacts with

Dsn1 and Nsl1 of the Mis12 complex (Petrovic et al., 2014). In addition to its function in outer

kinetochore assembly, Knl1 is also involved in spindle-assembly checkpoint signaling. Several

motifs have been identified within the disordered N-terminal region that are involved the

recruitment of signaling components. These include: i) the RVSF (RVXF) and the SILK motifs

which provide the binding site for the PP1 phosphatase (Liu et al., 2010), ii) the KI motifs (KI1

and KI2), which interact with the spindle assembly checkpoint (SAC) proteins (Bub1 and

BubR1) (Bolanos-Garcia et al., 2011; Kiyomitsu et al., 2011; Krenn et al., 2012) and iii) MELT

motifs, which act as mediators for other SAC proteins (Bub1 and Bub3) recruitment (London

et al., 2012; Shepperd et al., 2012; Yamagishi et al., 2012; Primorac et al., 2013). Thus, Knl1

acts as a central component for outer kinetochore assembly and faithful mitotic progression.

50

The third KMN subcomplex , the Mis12C is generally composed

of four subunits: Dsn1 (also identified as Knl3 in C. elegans

(Cheeseman et al., 2004)), Mis12, Nsl1 and Nnf1 (Goshima et

al., 1999, 2003; Cheeseman et al., 2004; Obuse et al., 2004;

Kline et al., 2006). An exception to this has been described in D.

melanogaster, which appears to have lost Dsn1 orthologue and

forms a tetrameric complex consisting of Mis12, Nsl1 and two

Nnf1 paralogs (Liu et al., 2016; Przewloka et al., 2007).

Depletion of Mis12C subunits in nematodes and human cells

leads to premature separation of spindle poles, misaligned and

lagging chromosomes (Figure bb), less compacted metaphase

plates and reduction of both SAC protein BubR1 and Ndc80C

(Goshima et al., 2003; Cheeseman et al., 2004; Kline et al.,

2006). Thus, the Mis12C is required for proper chromosome

segregation, chromosome alignment and spindle checkpoint

function. In addition, the depletion of one protein of the

subcomplex causes the loss of the other subunits at

kinetochore. Hence, Mis12C subunits are interdependent for

their kinetochore localization (Kline et al., 2006).

The Mis12C a 21-23 nm elongated rod structure (Petrovic et

al., 2010) acts as an interaction hub for the inner and outer kinetochore. The Mis12C interacts

directly with the N-terminal region of CENP-C (Przewloka et al., 2011; Richter et al., 2016;

Screpanti et al., 2011) and directly and indirectly (through the Ndc80C) with CENP-T

(Gascoigne et al., 2011; Nishino et al., 2013; Rago et al., 2015; Huis in ’t Veld et al., 2016). In

addition to these interactions with the inner kinetochore, the Mis12C binds both the Ndc80C

and Knl1 (Cheeseman et al., 2004; Obuse et al., 2004; Cheeseman et al., 2006; Petrovic et al.,

2010). Here, Nsl1 interacts with the C-terminal RWD domains of Knl1 (Petrovic et al., 2014)

and the RWD of Spc24 and Spc25 in a mutually-non-exclusive manner (Petrovic et al., 2010).

Thus, the Mis12C is important for the integrity of the KMN complex as a whole.

Figure bb. Mis12 complex is required

for accurate chromosome

segregation. Representative

immunofluorescence pictures of HeLa

Mis12C-depleted cells expressing

YFP-CenH3CENP-A. DNA is in blue and

tubulin in green. Depletion of the

Mis12C results in chromosome

alignment defects, while CenH3

centromeric localization is

unaffected. Scale bar: 10m. Figure

from Kline et al., 2006

51

Glimpses into kinetochore diversity

The discovery of kinetochore proteins and their role in chromosome segregation are mainly

derived from studies performed in a few model organisms such as yeast, worms, flies and

vertebrates (reviewed in Drinnenberg and Akiyoshi, 2017). These studies granted numerous

valuable information on kinetochore composition, assembly and function often conserved

across eukaryotes. For example, as described in the previous section, the complex set of the

CCAN in vertebrates and fungi has been identified and characterized due to large experimental

and computational efforts. While CCAN is largely

conserved across vertebrates and fungi, it appears

to be lost in flies and nematodes (Drinnenberg et al.,

2016) (Figure cc). These organisms appear to

contain a drastically reduced inner kinetochore

complex that only consists of CenH3 and its direct

DNA binding partner CENP-C. The functional

consequences on kinetochore function, if there are,

are currently unknown. In addition to studies

performed in established model organisms, large

scale homology prediction surveys across diverse

eukaryotic organisms and experimental analyses

performed in non-conventional model systems

revealed further insights into the plasticity of

kinetochore composition that stands in stark

contrast to its evolutionary conserved function

(reviewed in Drinnenberg and Akiyoshi, 2017).

To gain insights into kinetochore diversity across eukaryotes a recent study computationally

searched for homologs of kinetochore and spindle assembly checkpoint components across

well-assembled proteomes derived from organisms of all major eukaryotic groups (Hooff et

al., 2017) (Figure dd). To account for rapid protein sequence evolution of kinetochore

components, the authors applied remote homology prediction tools that consider both

sequence and structural information of the protein. This study revealed that subunits of a

single kinetochore complex, such as CCAN subcomplexes, co-evolve. In other words, two

Figure cc. Schematic model of kinetochores in A)

vertebrates, B) S. cerevisiae and C) D. melanogaster.

Kinetochore composition is highly similar between

vertebrates and fungi, with the exception of CENP-M

which is absent in budding yeast. In contrast to these

two types of kinetochores, in D. melanogaster the

majority of the CCAN components are lost. Figure

adapted from Drinnenberg et al., 2016

A) B)

C)

52

interacting kinetochore proteins are likely to be both conserved or both lost, thus they act as

a single functional unit. The exception to this pattern of co-evolution between CCAN

components are CENP-C, CENP-R and CENP-S/X, which might be explained by their functions

within the kinetochore complex. CENP-C is required for CenH3 nucleosome incorporation and

stabilization (Falk et al., 2015; Guo et al., 2017) and is sufficient to assemble part of the outer

kinetochore in flies and vertebrates (Gascoigne et al., 2011; Przewloka et al., 2011). CENP-S/X

are more ubiquitously conserved across evolution compared to other kinetochore proteins,

probably owed to their roles on DNA repair (Hooff et al., 2017). Finally, CENP-R appears to be

a more recent gene invention in animals (Hooff et al., 2017).

Intriguingly, this study also revealed that across evolution, several lineages lack kinetochore

components which are essential in conventional model organisms. These losses are probably

due to multiple independent gene loss events (Hooff et al., 2017) and raise the question of

how the kinetochore is assembled in absence of otherwise essential components. A notable

example for this is the loss of CenH3 (Figure dd).

CenH3 proteins have been identified in multiple lineages from fungi, plants and animals (Malik

and Henikoff, 2003). Despite the fact that these proteins are rapidly evolving, their

identification has been possible based on its structural conservation of the histone fold

domain. To distinguish among CenH3 homologs and other H3-like proteins several criteria

have been identified based on the protein sequence alignments of diverse CenH3 and H3

homologs. These criteria include: i) a highly divergent N-terminal tail, ii) a lack of a conserved

glutamine residue in the 1 helix, and iii) a longer loop 1 region compared to canonical H3

histone (Malik and Henikoff, 2003). Using these criteria together with phylogenetic

reconstructions CenH3 loss/absence has been identified in kinetoplastids, unicellular parasites

(Akiyoshi and Gull, 2014), an early-divergent fungus Mucor circinelloides (Hooff et al., 2017;

Navarro-Mendoza et al., 2019) and in multiple independent lineages of holocentric insects

(Drinnenberg et al., 2014).

53

Figure dd. Table summarizing the results of computational searches for kinetochore homologs in all major eukaryotic groups.

The presences are indicated as blue squares and absences as white squares. Top) Phylogenetic tree across 90 eukaryotic

lineages. Eukaryotic supergroups are color-coded. Notable, the authors identified that CenH3 is absent in early-divergent

fungus Mucor circinelloides (yellow arrow). Figure from Hooff et al., 2017.

In this section, I will briefly discuss the findings characterizing CenH3-deficient lineages.

Kinetoplastids: complete kinetochore rewiring

The kinetoplatids a group of unicellular flagellated eukaryotes have evolved a completely

different set of kinetochore proteins with no detectable homology to conventional

kinetochore components (Akiyoshi and Gull, 2014). Indeed, bioinformatic analyses failed to

identify CenH3 and any other kinetochore homolog while cell cycle components (e.g. Aurora

54

B, cohesin and condensin complexes, the CDK/cyclin system) were identified using this

method (Akiyoshi and Gull, 2013). Despite the lack of kinetochore protein homologs,

ultrastructure studies performed in the kinetoplastid Trypanosoma brucei (a parasite causing

the sleeping sickness) revealed kinetochore-like electron-dense plaques that form

attachments to spindle microtubules (Ogbadoyi et al., 2000). This result suggests that these

organisms segregate their chromosomes in a kinetochore-like dependent manner while

employing a completely different set of kinetochore components.

Recent studies based on proteomics and functional analyses in T. brucei uncovered that their

kinetochore is composed by 20 proteins called kinetoplastid kinetochore protein or KKT

(Akiyoshi and Gull, 2014; Nerusheva and Akiyoshi, 2016). These KKT proteins contain

functional domains (e.g. kinase domain, protein-protein interaction domains) and conserved

regions (e.g. coiled-coil, disordered regions) that have provided insights into their function

(reviewed in Akiyoshi, 2016) (Figure ee). For instance, both KKT2 and KKT3 have putative DNA

binding motifs (AT-hook and SPKK) suggesting that these components might bind DNA

(reviewed in: Akiyoshi, 2016; Drinnenberg and Akiyoshi, 2017). In addition to these, a recent

study revealed that KKT4 also binds DNA and has microtubule-binding domains. Thus far, KKT4

is the only kinetoplastid kinetochore protein known with microtubule-binding properties

(Ludzia et al., 2020). The function and structure of the majority of the KKT proteins remain to

be elucidated.

55

Figure ee. Kinetoplastid Trypanosoma brucei has evolved a completely different set of kinetochore proteins with no detectable

homology to conventional kinetochore components. Diagram of T. brucei KKT proteins and their identified domains and motifs.

Putative subcomplexes are clustered in dotted boxes. Figure from Akiyoshi and Gull et al., 2014.

Early divergent fungi: without CenH3 and CENP-C

Hooff et al. predicted the loss of CenH3 and CENP-C in early divergent fungi based on

computational analyses (Hooff et al., 2017) (Figure dd). More detailed predictions in addition

fungal species analyses confirmed that both CenH3 and CENP-C are indeed absent in the

Mucoromycotina clade but present in other fungi clades. Despite these losses, most of the

conventional kinetochore proteins continue to be present in these organisms (Navarro-

Mendoza et al., 2019). To follow up on these computational predictions, the Navarro et al

performed experimental analyses in Mucor circincelloides (member of the Mucoromycotina

clade that were found to lack CenH3 and CENP-C) to study its kinetochore composition and

centromere organization (Navarro-Mendoza et al., 2019). M. circincelloides is an opportunistic

human pathogen that causes an infectious disease called mucormycosis which is characterized

by rhino-orbital-cerebral, pulmonary and cutaneous infections, and it has high mortality rates

(Prakash and Chakrabarti, 2019). In contrast to other basal fungi which are difficult to

genetically manipulate, molecular tools are available for M. circincelloides (Zhang et al., 2016)

making it a potential model organism.

To study the localization of kinetochore components, the authors expressed fluorescent inner

(CENP-T) and outer kinetochore (Mis12 and Dsn1) proteins. Both Mis12 and Dsn1 displayed a

small fluorescent puncta signal in a single region that colocalized with chromatin and

fluorescently tagged CENP-T (Figure ff). These observations lead to the conclusion that despite

CenH3 and CENP-C losses, M. circincelloides kinetochores localize to a single

monocentromeric region of the chromosomes. Pulling down Mis12 and Dsn1 associated

centromeric DNA further revealed that M. circincelloides centromeres display features of both

point and regional centromeres (chapter 1). Indeed, M. circincelloides centromeres have short

kinetochore-binding regions, a DNA sequence motif specific to centromeres and an AT-rich

core region, features similar to point centromeres. On the other hand, the pericentromeres

are also associated with retrotransposons reminiscent to the regional chromosomes (Navarro-

Mendoza et al., 2019). Thus, the authors named them “mosaic centromeres”. Moreover,

56

other histone variants did not show centromere-

specific binding. Thus, it appears that CenH3

function at centromeric chromatin has not been

replaced by other histone variants (Navarro-

Mendoza et al., 2019). All together, these results

raise several questions that remains to be

addressed including: i) have other kinetochore

proteins replaced the function of CenH3 of

epigenetic marker and kinetochore foundation? ii)

is the centromere-specific DNA motif sufficient for

the centromere function?, and iii) is this motif the

binding site for an unknown centromere-specific

protein?

CenH3 has been lost multiple independent times in holocentric insects

Computational analyses across insects

revealed that multiple independent losses of

CenH3 in insects belonging to several orders

including: i) Lepidoptera (butterflies and

moths), ii) Hemiptera (true bugs)-

Phthiraptera (lice), iii) Dermaptera

(earwings) and iv) Odonata (dragonflies)

(Figure gg). Moreover, all these insects have

also undergone changes in their centromeric

architecture, in which each lineage

independently transitioned from

monocentric chromosomes to holocentric

chromosomes (chapter 1). In contrast,

Figure ff. M. circinelloides Mis12, Dsn1 and CENP-T

show discrete and puncta nuclear localization.

Pictures of live cell time-lapse of M. circinelloides

cells coexpressing: i)H3-eGFP (to identify chromatin)

with Mis12-mCherry (C) or Dsn1-mCherry (D), or ii)

coexpressing CENP-T-eGFP with Mis12-mCherry (E)

or Dsn1-mCherry (F). Mis12 and Dsn1-mCherry

displayed a small fluorescent puncta signal in a

single region that colocalized with the nucleus (H3-

GFP). Moreover, Mis12 and Dsn1 also colocalized

with CENP-T-GFP. Scale bar: m. Figure from

Navarro-Mendoza et al., 2019

Figure gg. Holocentric insects have lost CenH3. Phylogenetic

tree of insect orders. Holocentric insects are in blue and

monocentric insects in black. The black boxes indicate the

presence of CenH3 homologs and white/empty boxes indicate

the absence of CenH3 homologs. Figure from Drinnenberg et al.,

2014.

57

CenH3 homologs could be identified in monocentric insects including: Diptera (flies and

mosquitoes), Coleoptera (beetles), Hymenoptera (wasps, bees, and ants), Blattodea

(cockroaches), Phasmatodea (stick insects), Orthoptera (crickets) and Ephemeroptera

(mayflies) (Figure hh). Transcriptome assemblies of additional mono- and holocentric insects

further supported the presence of CenH3 in several monocentric insects, but its loss in several

holocentric insects. This observation was unlikely due to an insufficient coverage of the

transcriptome assembly nor to a lower CenH3 expression in holocentric insects compared to

monocentric ones. In fact, analyses revealed a comparable or even higher number of

predicted proteins in the holocentric assemblies supporting that transcriptome coverages

were comparable in both types of insects. Moreover, CenH3 transcripts were not in low

abundance in monocentric insects. Taking together, these findings showed that CenH3 was

lost multiple times in insects and that this loss is associated with the presence of holocentric

chromosomes (Drinnenberg et al.,

2014).

Interestingly, the authors found that

despite the absence of CenH3,

computational predictions revealed

that other CCAN components

continue to be present (Figure hh).

These include CENP-I, CENP-M, CENP-

L/N and CENP-S/X. The latter one’s

might also be conserved due to their

known roles in DNA repair.

Intriguingly, CENP-C, which in

vertebrates and fungi interacts with

CenH3 and the outer kinetochore,

was found to be absent in all insects

lacking CenH3 indicating that their

evolutionary presence is dependent

on one another. In contrast to the

inner kinetochore components,

Figure hh. Despite the CenH3 lost, other kinetochore proteins continue

to be present in holocentric insects. Holocentric insects are in blue and

monocentric insects in black. The black boxes indicate the presence of

homologs of conventional kinetochore proteins and white/empty boxes

indicate the absence of homologs of conventional kinetochore proteins.

Figure from Drinnenberg et al., 2014

58

homologs of the outer kinetochore proteins including the Ndc80C and the Mis12C, were

largely conserved in holocentric insects. Thus, despite the losses of CenH3 and CENP-C,

holocentric insects appear to have preserved the means to attach to the spindle microtubules.

Taken together, these discoveries suggest that holocentric insect species employ an

alternative kinetochore assembly mechanism that appears to occur chromosome-wide in a

CenH3-independent manner (Drinnenberg et al., 2014). While these findings have provided

some insights into the composition of CenH3-deficient kinetochores, key aspects of

kinetochore function, including how these kinetochores attach to centromeric chromatin and

to microtubules to drive cell division, remained unresolved.

To gain insights into these questions my host lab performed proteomic analyses of the CenH3-

deficient kinetochore in lepidopteran cell lines. For these analyses, computationally predicted

kinetochore components were pulled down to identify their interaction partners by mass

spectrometry. These studies identified a divergent homolog of CENP-T (for more details see

results section). Given its known role in DNA binding and outer kinetochore recruitment the

discovery of CENP-T in the CenH3-deficient kinetochore was of special interest to us to address

the key aspects on kinetochore assembly mentioned above.

59

Research aims

For my PhD project, I aimed to characterize kinetochore components and the assembly

cascade of the CenH3-deficient kinetochore in Lepidoptera. In particular, I focused on

understanding the role of CENP-T in Lepidoptera. In this context, I addressed the following

two key objectives:

(i) Characterize the dependency of CENP-T on other kinetochore components in the

CenH3-deficient kinetochore

(ii) Evaluate the role of CENP-T in outer kinetochore recruitment

To study CenH3-independent kinetochore assembly, I used cell lines from Bombyx mori

(BmN4) and Spodoptera frugiperda (Sf9). In order to address these aims, during the first and

second years of my PhD, I established molecular tools (stable cell lines, immunofluorescence

staining) and optimize protocols (RNAi-mediated depletion, characterization of mitotic

phenotypes) in these lepidopteran cell lines that are not commonly used for laboratory

research. Using those I gained insights into the following aspects on kinetochore assembly:

• The role of the identified kinetochore proteins in mitotic progression.

• The contribution of CCAN in CENP-T and outer kinetochore assembly.

• The contribution of CENP-T in outer kinetochore recruitment.

Taking together, the results of my PhD project will generate a novel model system for the

study of CenH3-independent kinetochore assembly. My findings will contribute to the

emerging picture of an unexpected plasticity in kinetochore composition and assembly across

eukaryotes.

60

61

Results

62

63

Identification of kinetochore components including CENP-T in CenH3-deficient Lepidoptera

To gain insights into the composition of CenH3-independent kinetochores, we performed

immunoprecipitation (IP) experiments of kinetochore components that we previously

identified in our computational survey (Drinnenberg et al., 2014) (Figure 1A). For this, we

established several stable Spodoptera frugiperda (Sf9) cell lines expressing 3xFLAG-tagged

CCAN (CENP-M, CENP-N, CENP-I) and outer kinetochore (Dsn1 and Nnf1) components to map

their protein interaction profiles by mass spectrometry.

Mass spectrometry analyses of the kinetochore immunoprecipitates recovered all of the

previously predicted homologs of outer kinetochore and inner kinetochore components,

confirming protein complex formation even in organisms that have lost CenH3 (Figure 1B,

Figure S1A). More importantly, our IPs also revealed several additional components, some of

which harbor remote homology to other known kinetochore components (Figure 1B). Among

those, we identified a CENP-K-like protein thereby experimentally supporting previous

homology predictions in Bombyx mori (Tromer, 2017). We also identified two components

(GSSPFG00019785001 and GSSPFG00001205001) with remote similarities to the coiled-coil

and RWD domains of the budding yeast CENP-O and CENP-P proteins, respectively.

Furthermore, we identified the homolog of the alpha subunit of the ATP synthase (bellwether)

(GSSPFG00010096001), previously found to localize to kinetochores during Drosophila

melanogaster male meiosis (Collins et al., 2018), indicating that this function might be

extended to lepidopteran mitosis.

Both inner and outer kinetochore immunoprecipitates also revealed the presence of a 191.2

kDa protein in S. frugiperda (GSSPFG00025035001) with KWMTBOMO06797 being the B. mori

homolog (Figure 1B). Iterative hidden Markov model (HMM) profile searches within

annotated proteomes first revealed homologs in several other insect orders and then known

vertebrate CENP-T homologs as best hits (Figure S2A). Similarly, HMM profile searches within

known protein structures identified the known Gallus gallus CENP-T as the best hit (Figure

S2B). The alignment between the lepidopteran and vertebrate CENP-T proteins was restricted

to their C-terminal parts that include the histone fold domain. Importantly, the alignment

extended to the known 2-helix CENP-T extension shown to interact with the CENP-H/I/K

64

complex in yeast (Pekgöz Altunkaya et al., 2016) with several conserved amino acids being

identical, supporting homology to CENP-T rather than to any other histone fold protein (Figure

1C, Figure S2C). The sequence aligning to the CENP-T extension appears to be even better

conserved than the HFD allowing for the identification of the G. gallus CENP-T in reciprocal

HMM profile searches (Figure S2B). Furthermore, analyses of the primary amino acid

sequence revealed additional similarities between the G. gallus CENP-T and the B. mori

protein including an enrichment of positively charged amino acids at the N-terminus and a

proline patch N-terminal of the HFD (Figure 1C). Finally, reciprocal IP experiment of the S.

frugiperda homolog confirmed its interaction with kinetochore components (Figure 1B).

Although orthology between the lepidopteran proteins and CENP-T cannot be confirmed

(Figure S2C), the common sequence architecture, kinetochore protein interaction and role in

outer kinetochore recruitment (see below) lead us to refer to this lepidopteran component as

CENP-T.

Interestingly, we could not detect a putative homolog of the CENP-T histone fold binding

partner CENP-W in any of our IP experiments including immunoprecipitates of a 3xFLAG-

tagged C-terminal truncated version of CENP-T that contains the HFD and 2-helix extension

(Figure S1B). Our ability to identify all other known kinetochore homologs in our kinetochore

IPs supports the idea that the inability to identify a lepidopteran CENP-W homolog is not due

to our IP protocol. However, limitations in detecting those potentially small, arginine-rich

proteins by mass spectrometry or incomplete annotations cannot be excluded.

Taken together, we have identified previously unknown kinetochore components in the

CenH3-deficient kinetochore of Lepidoptera. This includes putative homologs of known CCAN

components including CENP-T. These results reinforce and extend our previous computational

predictions indicating similar kinetochore components in Lepidoptera as those in vertebrates

and fungi, despite the loss of CenH3 in the former. Among those, the identification of CENP-T

was of particular interest due to its known function in DNA binding and outer kinetochore

recruitment in other organisms.

65

Figure 1. Identification of kinetochore components, including CENP-T, in CenH3-deficient Lepidoptera. A) Schematic

kinetochore organization in vertebrates and fungi. Boxes indicate subcomplexes within inner and outer kinetochores.

Kinetochore components present in Lepidoptera are highlighted in bold ( (Drinnenberg et al., 2014) this study). Kinetochore

components that cannot be identified or with limited homology are shown in gray. B) The table lists the number of peptides

and coverages (in parentheses) of known kinetochore homologs or S. frugiperda proteins identified by mass spectrometry that

were enriched in at least three of the kinetochore IPs over the control. Samples were boiled from the beads, and the analyses

were performed on the entire sample. The corresponding homologs in B. mori and descriptions based on homology predictions

are listed alongside. See also Figure S1. C) Top: graphical representation of the B. mori CENP-T and G. gallus CENP-T sequence

features, showing the location of the HFDs (blue box), CENP-T family- specific extension (green box), and arginine-rich (E value

4.7 x 10-5 and E value 6.0 x 10-7, respectively) and proline-rich regions (E value 3.7 x 10-4 and E value 3.4 x 10-8, respectively)

(orange and purple boxes, respectively). Bottom: multiple alignment of the C-terminal extension of vertebrate and insect

CENP-T proteins. B. mori (top) and G. gallus (bottom) secondary structure predictions are derived from Jpred4 (a-helical

regions are shown in red and b strands in yellow) (Drozdetskiy et al., 2015). Background coloring of the residues is based on

the ClustalX coloring scheme. See also Figure S2.Analyzes performed by I.A. Drinnenberg.

CENP-T and other kinetochore components are required for accurate mitotic progression

In other organisms, depletion of kinetochore components results in mitotic defects . Previous

studies in B. mori have shown that, consistently, depletion of outer kinetochore components

also results in mitotic defects and increased numbers of aneuploid cells (Mon et al., 2017). I

extended this previous study by using RNAi to deplete a broader catalogue of kinetochore

components. After some initial tests, I determined time points after induction of RNAi that are

A) B)

C)

66

suitable to characterize mitotic defects without extensive lethality of the cells. I depleted

several outer kinetochore components of the Mis12 complex (Dsn1, Mis12, Nsl1) and Ndc80

complex (Spc24 and Spc25) as well as several inner kinetochore components (CENP-T, CENP-

I, CENP-M, CENP-N). I studied the cells depleted for kinetochore components by

immunofluorescence (IF) at two time points (three and five days) (Figure 2A). Based on the

defects that I observed I categorized those into three types for quantitative analyses to

compare among different kinetochore depletion experiments. We further validated the

efficiency of our kinetochore mRNA depletions by RNA blot analyses (Figure S3A).

RNAi-mediated depletion of all kinetochore proteins tested resulted in an increase in the

number of mitotic cells relative to the control (RNAi targeting GFP), indicating that

kinetochore-depleted cells are delayed or arrested in mitosis. I observed the strongest

increase of mitotic cells upon CENP-T, CENP-I and outer kinetochore depletions while milder

enrichment was seen upon CENP-M and CENP-N depletion (Figure 2B). Interestingly, while

still above control levels, the mitotic indices upon CENP-I, Spc24 and Spc25 depletions were

strongly decreased at a later time point (Figure S3B), suggesting that possibly cells can exit

from mitosis and continue progressing through the cell cycle or undergo apoptosis.

In addition to the elevated mitotic indices, depletion of kinetochore components results in

various degrees of mitotic defects in these cells (Figure 2C, 2D, and Figure S3C, D, E and F).

Here, approximately one fourth of CENP-T depleted mitotic cells displayed misaligned,

monopolar chromosomes (where one or several chromosomes are not aligned at the

metaphase plate and remain at the spindle pole). In addition, 34% of CENP-T-depleted mitotic

cells showed complete failure of chromosomes to congress at the metaphase plate (Figure 2C

and 2D). This phenotype was even more pronounced upon CENP-I, Spc24 and Spc25

depletion, with the majority of cells (73%, 76% or 66%, respectively) unable to successfully

congress chromosomes (Figure 2C and 2D). In contrast, mitotic defects upon CENP-M and

CENP-N depletion (Figure 2C and 2D) were relatively mild but became more pronounced at

the later time point (Figures S3D, E and F). These results show that all tested inner and outer

kinetochore components are required for high fidelity chromosome segregation in B. mori

cells. Additionally, the defects observed upon depletion of the newly identified CENP-T further

support its role in chromosome segregation, which I aimed to analyze further.

67

Figure 2. Depletion of kinetochore components affects mitotic progression in B. mori cells. A) Schematic of the RNAi-mediated

depletion strategy. BmN4-SID1 (Mon et al., 2017) cell lines engineered for properties of enhanced uptake of dsRNA were

incubated with various dsRNA constructs for targeted depletion of kinetochore transcripts. After 3 and 5 days, cells were

analyzed by IF staining against anti-tubulin and anti-phospho histone H3-Ser10 (H3S10ph) in combination with each of our

custom-made antibodies against the kinetochore protein of interest. The growth medium was changed on day 3 to add new

dsRNA. The efficiency of kinetochore transcript depletion was confirmed by RNA blot analyses (Figure S3A). B) Graph showing

the percentage of mitotic cells (H3S10ph-positive cells) 3 days after RNAi-mediated depletion of targeted kinetochore

components (n = number of cells ± SEM). For results on day 5, see Figure S3B. C) Representative images of mitotic cells stained

with anti-tubulin (green), used to classify mitotic defects observed 3 days after depletion of select kinetochore components.

Scale bar, 10 m. For results on day 5 and depletion of additional outer kinetochore components, see Figures S3C and S3D.

For a zoomed-out version, see Figures S3E and S3F. D) Percentages of cells showing no defects (gray), monopolar

chromosomes (blue), congression defects (red), and both defects (combination, purple) (n = number of cells analyzed per

condition) for different kinetochore depletion experiments.

The CENP-T N- and HFD containing C-terminus are both essential for accurate mitosis

A)

B)

C)

D)

68

I next tested whether I can rescue CENP-T knock-down phenotypes by complementation with

a RNAi-resistant version of CENP-T. Using double-stranded RNA (dsRNA) targeting the

endogenous CENP-T transcript and a FLAG-tagged recoded CENP-T construct unable to be

targeted by the dsRNA, I was able to selectively deplete B. mori cells of endogenous CENP-T

but not FLAG-tagged CENP-T (Figure 3). These experiments were more qualitative than

quantitative due to the low transfection efficiency in lepidopteran cells in combination with

low numbers of mitotic cells that can be analyzed. Still, among the cells that could be analyzed,

expressing the full-length RNAi-resistant construct (CENP-Tres-FLAG) rescued the mitotic

defects described above (Figure 3). In contrast, cells expressing the wild-type, non-resistant

construct (CENP-T-FLAG) displayed mitotic defects as previously observed (Figure 2). To

evaluate whether the N- or HFD containing C-terminus of the CENP-T protein, or both, are

required for its function, I expressed RNAi-resistant FLAG-tagged N- and C-terminal truncated

CENP-T constructs (N-CENP-Tres-FLAG and C-CENP-Tres-FLAG). Cells expressing either of

the two constructs failed to rescue the observed mitotic defects (Figure 3), leading to the

conclusion that the full-length CENP-T protein is necessary for accurate mitosis.

A)

B)

69

Accurate localization of CENP-T is dependent on other inner kinetochore components and

is necessary to recruit the Mis12 complex

Next, to evaluate the role of CCAN and outer kinetochore components in kinetochore

assembly I depleted individual kinetochore proteins and stained with custom made antibodies

against the lepidopteran CENP-T, Dsn1 (Mis12 complex) and the Spc24/Spc25 (Ndc80

complex). We validated the specificity of the antibodies by mass spectrometry, western blot

analyses and IF upon RNAi-mediated depletion of the respective genes (Figure 4A, Figure S4).

As expected for kinetochore components, I observed the immunosignals of CENP-T, Dsn1 and

Spc24/Spc25 co-localizing with mitotic chromosomes (H3S10ph positive cells) in control BmN4

cells (RNAi targeting GFP) (Figure 4A). Then, as automated systems failed to detect the nuclear

areas, I manually selected them to quantify the intensity of the immunosignals on mitotic

chromosomes in control and kinetochore depleted cells (Figure 4B). Depletion of CENP-I,

CENP-M and CENP-N significantly impaired CENP-T localization to mitotic chromatin. In

contrast, in cells depleted for outer kinetochore components (Dsn1, Mis12, Nsl1, Spc24 and

Spc25) the CENP-T immunosignal could still be observed (Figure 4B and 4C). In addition,

depletion of any CCAN component resulted in the loss of Dsn1. In contrast, the recruitment of

Spc24/Spc25, while reduced upon other CCAN depletion, was only completely abolished upon

depletion of CENP-I (Figure 4B and 4C), consistent with its severe chromosome alignment

defects (Figure 2). Dsn1 and Spc24/Spc25 localization was also abolished in cells depleted of

other members of their respective complexes (Figure 4B and 4C, Figure S4). These data

suggest that in B. mori, the recruitment of CENP-T is dependent on other inner kinetochore

components. In turn, CENP-T appears to be required to recruit the Mis12 complex.

Figure 3. The B. mori CENP-T N- and HFD containing C-terminus are both essential for mitotic progression. A) Schematic

representation of the wild-type FLAG-tagged CENP-T (CENP-T-FLAG), FLAG-tagged RNAi-resistant CENP-T (CENP-Tres-FLAG),

the N-terminal (ΔN-CENP-Tres-FLAG) and C-terminal truncated RNAi-resistant CENP-T (ΔC-CENP-Tres-FLAG). The recoded

region conferring RNAi-resistance to the used dsRNA against CENP-T is indicated in orange (see Methods). B) Representative

IF images of mitotic cells(n= 3 cells analyzed) expressing CENP-T-FLAG (first row) followed by RNAi-mediated depletion of

endogenous and exogenous CENP-T. Representative IF images of cells expressing full-length CENP-Tres-FLAG (second row),

ΔN-CENP-Tres-FLAG (third row) or ΔC-CENP-Tres-FLAG (fourth row) RNAi-resistant FLAG-tagged CENP-T followed by RNAi-

mediated depletion of endogenous CENP-T. Scale bar: 10μm.

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Figure 4. Depletion of CCAN components affects CENP-T and outer kinetochore recruitment in B. mori cells. A) Representative

images of mitotic BmN4-SID1 cells, showing the levels of endogenous CENP-T, Dsn1, and Spc24/Spc25 with and without

depletion of inner (CENP-T, CENP-I, CENP-M, and CENP-N) and outer (Dsn1 and Spc24) kinetochore components. Scale bar, 10

m. B) Quantification of mean fluorescence intensity for CENP-T, Dsn1, and Spc24/25 signals in the control or upon

kinetochore depletion. Statistical significance was tested using a Mann-Whitney test (****p % 0.0001, ***p % 0.001, **p %

0.01, *p % 0.05, ns: not significant). For depletion of additional outer kinetochore components, see Figure S3. C) Table

A)

B)

C)

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summarizing the centromeric localization results of endogenous CENP-T, Dsn1, and Spc24/25 upon RNAi depletion. ++

represents control levels, + represents reduced fluorescence levels compared with control levels, and – refers to undetected

levels. See also Figures 2 and S3.

Targeting CENP-T to ectopic sites recruits the Ndc80 and Mis12 outer kinetochore complexes

My next aim was to evaluate whether CENP-T is sufficient to recruit outer kinetochore

components. For this, I first optimized co-transfection condition in B. mori cells to enable the

uptake of two plasmids by the cells. In particular, I transfected B. mori cells with a plasmid

containing Lac operator (LacO) arrays, which enables the targeting of transiently expressed

CENP-T-GFP-LacI protein or control (LacI-GFP) constructs to these loci. To test for the presence

of kinetochore components I stained the cells with our custom-made antibodies against the

N-terminus of CENP-T, Dsn1 and Spc24/Spc25. I selected equal-sized areas over the GFP foci

and over mitotic chromosomes and calculated the ratio of their immunosignals to determine

recruitment of kinetochore proteins to the LacO array.

As expected I observed elevated CENP-T immunosignals over the GFP foci compared to

endogenous loci in cells expressing full-length B. mori CENP-T-GFP-LacI. In addition, I also

observed elevated Dsn1 and Spc24/Spc25 immunosignals over the GFP foci indicating that

CENP-T is capable to recruit both the Mis12 and Ndc80 outer kinetochore complexes. In

contrast, in cells expressing LacI-GFP neither CENP-T, Dsn1 nor Spc24/Spc25 immunosignals

are enriched over the LacI-GFP foci (Figure 5).

In cells expressing the N-CENP-T-GFP-LacI fusion protein that is unable to be recognized by

our CENP-T antibody (Figure S4E) I did not measure elevated CENP-T immunosignals over the

GFP foci indicating that endogenous CENP-T is not recruited to the LacO array. The ratios of

Dsn1 and Spc24/Spc25 immunosignals were reduced compared to those in cells expressing

the full-length CENP-T fusion proteins, which indicates that the N-terminus of CENP-T

contributes to the recruitment of these outer kinetochore complexes (Figure 5). Taken

together, I conclude that CENP-T is sufficient for the recruitment of the Ndc80 and Mis12 outer

kinetochore complexes and its N-terminus appears to be important for this activity.

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Figure 5. Ectopic CENP-T is sufficient to recruit the outer kinetochore in B. mori cells. A) Representative images showing

localization patterns of endogenous CENP-T, Dsn1, and Spc24/Spc25 (gray) in BmN4-LacO cells transiently expressing LacI-

GFP (top row), CENP-T-GFP-LacI (center row), and N-CENP-T-GFP-LacI (bottom row) constructs (green). Scale bars, 10 m.

B) Quantifications of mean fluorescence intensity of CENP-T, Dsn1, and Spc24/Spc25 at GFP foci versus CENP-T, Dsn1, and

Spc24/Spc25 at endogenous sites (n = 10 cells analyzed). The ratios of mean fluorescence intensities on GFP foci over

endogenous loci are shown. Statistical significance was tested using a Mann- Whitney test (****p % 0.0001, **p % 0.01, *p

% 0.05, ns: not significant). See also Figure S4.

CENP-T is essential in vivo

We next aimed to evaluate the importance of CENP-T in vivo. For this, CRISPR/Cas9-mediated

gene editing was applied to introduce mutations into the endogenous CENP-T gene of the B.

mori N4 reference strain. A mutant (+7) was isolated that contains a 7-base pair insertion

within the guide RNA target site causing a pre-mature STOP codon in the CENP-T gene (Figure

6A). Since the C-terminus of B. mori CENP-T containing the HFD and CENP-T extension appears

to be essential for its function (Figure 3), the premature stop codon will lead to a non-

A)

B)

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functional protein product. Heterozygous CENP-T mutants can be readily propagated but

when crossed to each other the proportion of unhatched eggs increased to about 30%

compared to control crosses (Figure 6B). Genotypic analyses of the progeny of this cross

coming from 70% eggs that hatched did not reveal any homozygous CENP-T mutants (Figure

6C). These results show that as in vitro, CENP-T is also essential for B. mori in vivo.

Figure 6. A) CRISPR/Cas9-introduced mutation in the Cenp-T coding sequence. Top: alignment between wild-type (WT) and

mutant (+7) sequences, showing a 7-bp insertion of the Cenp-T gene. Bottom: schematic of WT and truncated protein products

in the CRISPR mutant. The PAM site (red), premature stop codon (blue), and Cas9 cleavage site (red arrow) are indicated. B)

Hatching rate of the WT and Cenp-T mutant strains. The percentages of hatched (gray), unhatched (green), and eggs that

died prior to hatching (black) are indicated for the different crossing patterns between WT (+/+) and heterozygous Cenp-T

mutants (+/knockout [KO] or KO/+). The number of independent crosses (n) is shown above. The raw data are shown in Table

S1. C) Percentages of genotypes from larvae derived from the cross between two heterozygous Cenp-T mutants. The number

(n) of analyzed larvae is indicated. The raw data are shown in Table S2. Experiment and analyses performed by T. Kiuchi and

S. Katsuma.

Homologs of CENP-T are retained in independently derived CenH3-deficient insects

Having characterized the function and importance of CENP-T for CenH3-independent

kinetochore formation in Lepidoptera, we next profiled its conservation across other CenH3-

deficient and CenH3-encoding insects. Orthologs of the lepidopteran CENP-T protein can be

A)

B) C)

74

readily identified using BLASTP in all CenH3-deficient insects analyzed as well as several

CenH3-encoding insects (Figure 7). Notably, phylogenetic analyses of HFD proteins belonging

to various HFD families including CENP-T indicated accelerated rates of CENP-T protein

sequence evolution ancestral to all insects (Figure S2C). Importantly, the retention of CENP-T

homologs in independently derived CenH3-deficient insects orders indicates important roles

for CENP-T in kinetochores in these organisms.

Preliminary analyses show the ability of CENP-I to recruit CENP-T and the outer kinetochore

At the end of my PhD and following the publication of my PhD paper, I generated preliminary

data to further understand the role of CENP-I. This protein was particular interesting to us

given its severe mitotic defects upon its depletion. Furthermore, CENP-I also appeared to be

the only component upon which depletion the recruitment of the Ndc80 outer kinetochore

complex was completely abolished. In preliminary experiments that I performed towards the

end of my PhD, I thus evaluated whether CENP-I is sufficient to recruit the Mis12 and the

Figure 7. Homologs of CENP-T are present in all other CenH3-deficient insects. Holocentric insect orders and species are

indicated in blue, and inferred multiple transitions to holocentric chromosomes are labeled with ‘‘H.’’ Using protein

homology searches of genomes or assembled transcriptomes, the ability and inability to find CenH3 and CENP-T homologs

are indicated by a black or white box, respectively. Although our previous studies did not identify any holocentric insect

species that encode for CenH3 (Drinnenberg et al., 2014), searches in additional organisms revealed the presence of putative

CenH3 proteins in water striders (Hemiptera). This result provides new insights into the transition to CenH3-deficient

holocentric architectures in that the change in centromeric architecture appears to precede the loss of CenH3, perhaps by

providing the necessary conditions to allow loss of this otherwise essential component. Analyses performed by I.A.

Drinnenberg.

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Ndc80 complexes, and even CENP-T. For this propose, I artificially tethered LacI-GFP-CENP-I

to LacO arrays. As for the CENP-T artificial tethering experiments, I tested for the presence of

CENP-T, Dsn1 and Spc24/25 using our custom-made antibodies (Figure 8). I found that the

immunosignals of all these proteins were elevated over the GFP foci, indicating that CENP-I is

sufficient to recruit both the Mis12 and the Ndc80 complexes, and CENP-T. To further

determine which CENP-I region might be responsible of these recruitments, I expressed both

the LacI-GFP-N-CENP-I and the LacI-GFP-CENP-I-C fusion proteins in LacO-containing cells

(Figure 8). Here, I found that both truncated fusion proteins failed to recruit CENP-T, Dsn1 and

Spc24/Spc25 proteins. To what extend these truncated versions of CENP-I are able to integrate

into the CenH3-independent kinetochore is however unclear.

BmQuant_LacOCENP-

I_17022020.xlsxBmQuant_LacOCENP-

I_17022020.xlsxBmQuant_LacOCENP-

I_17022020.xlsxBmQuant_LacOCENP-

I_17022020.xlsx

CENP-T

CENP-T Dsn1 Spc24/25CENP-T

A)

B)

LacI-GFP-D NCENP-I

D N

D N-CENP-I

LacI-GFP-CENP-ID C

D C

CENP-I- D C

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Figure 8. Ectopic CENP-I is sufficient to recruit the endogenous CENP-T and the outer kinetochore in B. mori cells. A)

Representative images showing localization patterns of endogenous CENP-T, Dsn1, and Spc24/Spc25 (gray) in BmN4-LacO

cells transiently expressing LacI- GFP (top row), LacI-GFP-CENP-I (2nd row), LacI-GFP-NCENP-I (3rd row) and LacI-GFP-CENP-

IC (bottom row) constructs (green). Scale bar: 10 m.B) Quantifications of mean fluorescence intensity of CENP-T, Dsn1, and

Spc24/Spc25 at GFP foci versus CENP-T, Dsn1, and Spc24/Spc25 at endogenous sites (n = 10 cells analyzed). The ratios of mean

fluorescence intensities on GFP foci over endogenous loci are shown. Statistical significance was tested using a Mann- Whitney

test (****p % 0.0001, ***p % 0.001, **p % 0.01, *p % 0.05, ns: not significant).

Analyses towards the localization dynamics of CENP-T and CENP-I

As described in the introduction, CenH3 epigenetically marks centromeres in most eukaryotes.

To accomplish this function, CenH3 remains stably associated with chromatin (Régnier et al.,

2005; Jansen et al., 2007). A stable association with chromatin is therefore a feature expected

for an epigenetic marker.

During the first year of my PhD I studied the localization dynamics of CENP-T and CENP-I to

test whether one or both components stably associate with chromatin. This would support

the possibility that either one or both epigenetically mark centromeres in Lepidoptera in the

absence of CenH3. Using our BmN4 cells, I used fluorescence recovery after photobleaching

(FRAP) assays (van Royen et al., 2008) to determine the dynamics of CENP-T and CENP-I over

shorter time scales. This approach has previously been used to measure the dynamics of

components in CenH3-dependent kinetochore complexes in human cells (Hemmerich et al.,

2008). The authors report that the majority of kinetochore proteins are stable during mitosis

but dynamic during interphase. The notable exception to this was CenH3 which remains stable

through interphase consistent with its important role in epigenetically marking centromeres

location.

Similarly, I have expressed kinetochore GFP fusion proteins, then photo-destroyed the GFP

signal in a particular spot and measured the time required to repopulate the bleached area. If

there is a fast recovery of the fluorescent signal in the bleached area, this would suggest that

the proteins studied are dynamic. On the other hand, if no recovery of the fluorescent signal

is observed, this suggests that those proteins are stably bound to chromatin.

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In a first instance, I tried to express CENP-T and CENP-I GFP-tagged proteins driven by their

endogenous promoters (previously isolated from genomic DNA and tested). However, the low

fluorescent signal made impossible to bleach and study the possible fluorescent signal

recovery under these conditions. To overcome these technical problems, I decided to express

these fusion proteins under the stronger baculovirus promoter in order to increase the signal

to background ratio. I found that both proteins, CENP-T-GFP and CENP-I-GFP, quickly

repopulated the bleached area during interphase, suggesting that they are highly dynamic.

However, when the bleach was done during mitosis, these proteins failed to repopulate the

bleached area (Figure 9). I therefore conclude that ectopic CENP-T and CENP-I fusion

constructs are dynamic during interphase but stably bound to chromatin during mitosis. Such

dynamic behavior of the lepidopteran CENP-T and CENP-I proteins during interphase would

be inconsistent with those acting to epigenetically mark centromere location.

A)

B)

C)

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Figure 9. CENP-T-GFP and CENP-I-GFP are dynamic in interphase and stably bound to chromatin during mitosis. (A) Schematic

for FRAP assay. The GFP signal will be photo-destroyed to later measure the recovery time to evaluate the dynamics and

stability of kinetochore components CENP-T-GFP and CENP-I-GFP. (B) Representative pictures for CENP-T-GFP (metaphase:

top left, interphase :top right) and CENP-I-GFP (metaphase: bottom left, interphase :bottom right). Bleached area (red square).

Scale bar : 25μm (C) Mean FRAP recovery curves of CENP-T-GFP (top left, n=2 cells analyzed) and CENP-IGFP (bottom left, n=2

cells analyzed) in mitosis show no recovery after bleaching, but during interphase CENP-T-GFP (top right, n=10 cells analyzed)

and CENP-I-GFP (bottom left, n=9 cells analyzed) show a fast recovery of fluorescent signal, around 2 sec, during interphase.

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Discussion The results of my PhD project provide new insights into the plasticity of kinetochore

formation. CenH3 has long been thought to be the cornerstone of kinetochore formation by

mediating the attachment of the kinetochore to chromatin. Its direct DNA binding partner

CENP-C in turn contributes to the assembly of other CCAN components and the recruitment

of the outer kinetochore in mitosis (Carroll et al., 2010; Gascoigne et al., 2011; Przewloka et

al., 2011; Screpanti et al., 2011; Kato et al., 2013; Klare et al., 2015). In addition to these two

central components, other conserved CCAN components including CENP-T that I mainly

focused on in this thesis, emerged as another core component of the kinetochore capable of

bridging chromatin to the outer kinetochore complex in vertebrates and fungi (Hori et al.,

2008a; Schleiffer et al., 2012; Nishino et al., 2013). The discovery and characterization of

CENP-T in addition to the analyses of other CCAN components in CenH3-deficient Lepidoptera

provides the first insights into alternative pathways to build a CCAN-based inner kinetochore

in a CenH3-independent manner.

My assays using CENP-T artificial tethering indicate that the role of the lepidopteran CENP-T

in outer kinetochore recruitment might be similar to that in vertebrates and fungi. As

described in the introduction, in vertebrates, CENP-T is sufficient for the recruitment of both

the Mis12 and the Ndc80 complexes by directly interacting with their respective subunits

(Gascoigne et al., 2011; Hara et al., 2018; Huis in ’t Veld et al., 2016). In fungi, the CENP-T N-

terminal directly interacts with the Spc24/Spc25

proteins to recruit the Ndc80 complex (Malvezzi et al.,

2013; Schleiffer et al., 2012). Our results show that the

lepidopteran CENP-T is also sufficient to recruit the

Ndc80 and Mis12 complexes (Figure 5 and Figure D1).

Whether or not CENP-T, in particular its N-terminus

makes direct protein interactions with any of the Ndc80

or Mis12 complex subunits remains to be shown.

Moreover, CENP-T might not be the only factor

recruiting outer kinetochore complexes. In fact, though strongly reduced, Spc24/Spc25 and

Dsn1 are still present on N-CENP-T-GFP-LacI foci, which indicates that their recruitment is

Figure D1. Schematic of lepidopteran

kinetochore subunits analyzed in this study.

Black arrows indicate full localization

dependencies. Grey dashed arrows indicate that

localization dependency is unknown.

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either aided by more internal regions of CENP-T or via another kinetochore components

recruited by CENP-T. Furthermore, I observe that the Spc24/25 recruitment is strongly

reduced but not completely abolished upon CENP-T depletion (Figure 4 and Figure D1). This

result suggests that it exists at least one additional Ndc80 receptor at the kinetochore,

perhaps via CENP-I.

In addition to my results on CENP-T, my studies revealed that other CCAN components also

appear to have essential roles in the assembly of CenH3-deficient kinetochores. For instance,

depletions of CENP-M, CENP-N and CENP-I result in the absence of the Mis12 complex

recruitment at centromeres. In contrast, the recruitment of the Ndc80 complex is only

completely abolish upon CENP-I depletion. I thus chose to follow-up my functional analyses

on CENP-I using the tools that I have established. In my preliminary data using the LacI-LacO

tethering system, I could show that CENP-I (as CENP-T) is even sufficient to recruit the Mis12

and Ndc80 outer kinetochore complexes (Figure 8). A role of the lepidopteran CENP-I in

recruiting the Ndc80 complex could recapitulate previous observations in other organisms

showing that the vertebrate CENP-H/I/K/(M) complex contributes to Ndc80 localization

(Cheeseman et al., 2008). Indeed, the C-terminus of human CENP-I localizes closely to Ndc80

(Suzuki et al., 2015) and the CENP-H/I/K/(M) subunits directly interact with Ndc80 in

vertebrates and budding yeast (Mikami et al., 2005; Pekgöz Altunkaya et al., 2016).

Nevertheless, as it is the case for CENP-T, whether or not the lepidopteran CENP-I directly

interacts with any outer kinetochore complexes remains to be shown.

Beyond the characterizations of CENP-T and CENP-I in this thesis, future studies using the LacO

tethering system will aim to get further insights into the localization dependencies of inner

kinetochore components and their relevance to recruit the outer kinetochore. Indeed, my

preliminary data already contribute to this question showing that CENP-I is capable to recruit

CENP-T (Figure 8). Moreover, the role of other CCAN as CENP-M, CENP-N or the newly

identified CENP-K can be evaluated in Dsn1, Spc24/25 and CENP-T recruitment. It would also

be interesting to evaluate whether the recruitment of CENP-T and other inner kinetochore

components are mutually exclusive on one another. Given the unavailability of antibodies

against other inner kinetochore components except CENP-T, their recruitment to the tethered

component will be studied using ectopically expressed inner kinetochore GFP fusion proteins.

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In addition, RNAi-mediated depletions of kinetochore components can be used to evaluate

their contribution as mediators involved in kinetochore recruitment. For example, we could

evaluate if LacI-GFP-CENP-I continues to recruit endogenous CENP-T in CENP-M-depleted

cells. Overall, the tools that were generated in this study will facilitate future studies to dissect

the contribution of CENP-I, other CCAN, or additional kinetochore components with unknown

evolutionary relationships to kinetochore assembly in Lepidoptera (Figure 4).

Considering the essential roles of CenH3 and CENP-C in all other organisms tested, the

recurrent loss of these proteins in several insects indicates that the potential for CenH3/CENP-

C-independent kinetochore formation might have already arisen in an early insect ancestor.

Such potential could be represented in the form of other kinetochore components that

evolved the capability to compensate (partially or completely) for CenH3/CENP-C-dependent

roles in kinetochore-chromatin attachment and outer kinetochore recruitment. Notably,

given that D. melanogaster has lost most CCAN components and solely relies on CenH3 and

CENP-C for inner kinetochore assembly, it is unlikely that this species can lose either one of

these two proteins. The retention of CENP-T homologs in independently-derived

CenH3/CENP-C-deficient insects (Figure 7) however suggests their important contributions to

kinetochore assembly, perhaps by contributing CenH3/CENP-C mediated functions. Among

these, future studies will in particular aim to evaluate the contribution of the lepidopteran

CENP-T in kinetochore-chromatin attachment. For this, it will be important to better

understand the composition of the CENP-T containing complex.

As stated above, we were unable to identify a potential candidate of CENP-W in Lepidoptera

perhaps due to limitations in detecting potentially small, arginine-rich proteins (as CENP-W)

by mass spectrometry or incomplete annotations in S. frugiperda proteome (Figure 1).

However, there are also other possibilities that could explain our inability to detect a

lepidopteran CENP-W homolog. One of those is that CENP-T may not have a binding partner,

this scenario is however unlikely due to the hydrophobic nature of the dimerization interphase

of HFD-containing proteins. Instead, it is possible that the CENP-T binding partner is in fact a

canonical histone. Finally, homodimerization of CENP-T via its HFD could also be considered

as another possible scenario. Jonathan Ulmer, a biochemist of the lab, tested this possibility

by purifying the S. frugiperda HFD of CENP-T expressed in Sf9 cells. He then performed size

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exclusion chromatography with multiangle light scattering (SEC-MALS) but found no evidence

for dimerization of CENP-T. The estimated molecular weight by this analysis corresponded to

the one expected for the monomeric protein fragment. Nevertheless, limitation in detecting

CENP-T dimerization due to our protocol cannot be excluded. Intriguingly, the purified S.

frugiperda HFD of CENP-T was soluble, which is unexpected given HFD (due to its hydrophobic

nature, see above). In order to better understand the biochemical behavior of the

lepidopteran CENP-T, future studies determining its structure could provide better insights.

Nevertheless, it is also possible that CenH3/CENP-C-mediated functions have been

compensated by other CCAN components. In fact, several other CCAN components including

CENP-I and CENP-N are also retained in CenH3-deficient insects (Drinnenberg et al., 2014)

indicating critical roles in their kinetochore assemblies as well. While my tethering approach

provides insights into the assembly cascade of the kinetochore, this approach does not

address the question of how the kinetochore attaches to chromatin or DNA. For this,

DNA/chromatin binding activity of CENP-T, CENP-I, CENP-N and other kinetochore

components could be evaluated by expressing those in heterologous systems such as S2 cells,

testing for the presence of DNA in their immunoprecipitates or using in vitro analyses. CENP-

N will be particularly interesting to follow-up in this respect, given its known capability of

chromatin binding, preferentially CenH3-containing chromatin in other organisms (McKinley

et al., 2015; Pentakota et al., 2017). Given the loss of CenH3, it will be interesting to test

whether the lepidopteran CENP-N evolved to prefer binding to H3- over CenH3-containing

nucleosomes instead. DNA/chromatin binding activity is a necessary feature to initiate de

novo centromere formation, thus helping to identify the most upstream component for

CenH3-independent kinetochore assembly.

The absence of CenH3, known to epigenetically mark centromeres in most eukaryotes (see

introduction) also raises the question whether those organisms use alternative mechanisms

or components to epigenetically mark their centromeres. A recent study from our lab mapping

and characterizing centromeric regions along B. mori holocentric chromosomes support the

hypothesis that this might not be the case (Senaratne et al., 2020). In this study, centromeres

distribution positively correlated with transcriptionally silenced chromatin, and anti-

correlated with active histone modifications, nucleosome turnover and RNA polymerase II.

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These observations led to the hypothesis that B. mori centromeres appear to be shaped by

the chromatin environment. Furthermore, transcriptional perturbation experiments showed

that indeed centromeres are excluded from active chromatin regions but can be re-

established in the same regions when chromatin activity is again low. We thus currently favor

the model that in B. mori, the centromeres localization is dependent on the chromosome-

wide chromatin landscape, rather than an active epigenetic mechanism that allows the

inheritance of centromeric loci. This model would also predict that none of the kinetochore

components stably associates with chromatin. Indeed, as described earlier, stable chromatin

association has been shown to be a distinguishing feature of CenH3, which is important for

the epigenetic inheritance of centromere location in other organisms (Régnier et al., 2005;

Jansen et al., 2007).

My preliminary data on CENP-T and CENP-I dynamics are consistent with our model for

centromere specification in B. mori. (Figure 9). Neither one of these two proteins appear to

be stably associated with chromatin during interphase. Nevertheless, I hesitate to draw a

strong conclusion of these results given the fact that I needed to overexpress these

components in order to perform my experiments. While I attempted to do so in my first

assays, future studies could aim to study the dynamics of CENP-T and CENP-I expressed at

their endogenous levels. In addition, it will also be interesting to study the dynamics of other

kinetochore components including CENP-N. In fact, the possibility that another

uncharacterized kinetochore protein may act as centromere epigenetic marker in some

regions of the genome cannot be completely excluded.

Taken together, our results exemplify the necessity of experimental data to obtain

comprehensive pictures of kinetochore complex composition. The characterization of

kinetochore components in additional eukaryotes will enable us to obtain better insights into

the sequence divergence of kinetochore homologs. This will allow us to increase the

information content of our alignments thereby improving our homology prediction

capabilities. Finally, our studies on kinetochore composition of CenH3-deficient holocentric

lepidopteran species, together with the recent finding of a CenH3-deficient monocentric

fungus (Hooff et al., 2017; Navarro-Mendoza et al., 2019) that encodes for other CCAN

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components including CENP-T, show that CCAN-based CenH3 independent kinetochore

assemblies are considerably widespread in diverse eukaryotes.

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Methods

86

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EXPERIMENTAL MODEL AND SUBJECT DETAILS

Lepidopteran cell lines and culture conditions

Cultured silkworm ovary-derived BmN4 (ATCC Cat#CRL-8910; RRID: CVCL_Z633) and BmN4-

SID1 cell lines (RRID:CVCL_Z091) (Kobayashi et al., 2012) were maintained in Sf-900 II SFM

medium (Gibco Cat#10902-088) supplemented with 10% fetal bovine serum (Eurobio

Cat#CVFSVF0001), antibiotic-antimycotic (Gibco Cat#15240-062) and 2mM L-glutamine

(Gibco Cat#25030-024) at 27 °C. Sf9 cells (Gibco Cat#12659017) were maintained in Sf-900 II

SFM medium (Gibco Cat#10902-088) supplemented with antibiotic-antimycotic (Gibco

Cat#15240-062) and 2mM L-glutamine (Gibco Cat#25030-024) at 27 °C.

Culture conditions of B. mori N4 strain

Non-diapaused larvae of the B. mori N4 strain were fed with fresh mulberry leaves or artificial

diet SilkMate (NOSAN SilkMate PS) under a continuous cycle of 12-h light and 12-h darkness

at 25°C.

METHOD DETAILS

Alignments and phylogenetic analyses

For Figure 1C and Figure S2C, sequences were aligned with MAFFT on the EMBL-EBI web

interface (Madeira et al., 2019), and visualized and processed with Jalview (Waterhouse et

al., 2009) (ClustalX colouring scheme). Secondary structures were predicted using Jpred4

(Drozdetskiy et al., 2015). For Figure S2D, histone fold domains were aligned using MAFFT

(Katoh and Standley, 2013; Lemoine et al., 2019). A maximum likelihood phylogeny was build

using PhyML 3.0 (Guindon et al., 2010) with automatic model selection (Lefort et al., 2017),

default parameters and 100 bootstrap permutations. The tree was visualized using iTOL vs4

(Letunic and Bork, 2019). Primary sequence analyses of the B. mori and G. gallus CENP-T

(NP_001263242.1) for Figure 1C were performed using fLPS (Harrison, 2017) with default

parameters. The Arginine-rich N-terminal regions are located between amino acid 4 and 32 of

the G. gallus CENP-T (E value 6.0x10-7); and 20 and 41 of the B. mori CENP-T (E value 4.7x10-

5). The proline-rich regions are located between amino acid 493 and 521 of the G. gallus CENP-

T (E value 3.4x10-8); and 850 and 899 of the B. mori CENP-T (E value 3.7x10-4).

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Homology predictions

The annotation of the S. frugiperda corn strain proteome were used for all computational

analyses (Gouin et al., 2017). In case annotations were missing or incorrect we complemented

the data using annotations from the S. frugiperda “rice strain” (Gouin et al., 2017), EST

(Transcriptome TR2012b) data (Nègre et al., 2006) or our own analyses. Homologs of S.

frugiperda proteins identified in the kinetochore IPs were predicted in B. mori proteome

(Kawamoto et al., 2019). Both S. frugiperda and B. mori proteins were used for homology

searches. BLASTP and PSI-BLAST searches were performed in the NCBI non-redundant protein

database (Sayers et al., 2019). Jackhmmer searches were performed on the HMMER

webserver (Potter et al., 2018) against reference proteomes as current as September 2019.

HHpred version 3.2.0 searches were performed against the PDB_mmCIF70_3_Aug

(Zimmermann et al., 2018). Coiled-Coil domains were predicted using PCOILS (window 28)

(Jones, 1999; Gruber et al., 2006; Zimmermann et al., 2018).

CENP-T

For iterative HMM profile searches only the C-termini containing the HFD and 2 helix

extension of the B. mori or S. frugiperda proteins were used.

>B_mori_CENPT_CTERM

TTKRLYKYLEDKLEPKYDYKARVRAEKLVETIYHFTKEVKKHEVAPNDAVDVLKHEMARLDI

VKTHFDFYQFFHDFMPREIRVKVVPDIVNKITIPRNGVFSEILSGHAVHA

>S_frugiperda_CENPT_CTERM ITKRLYKFLETKLEPKYDYKARVRAEKLVETIYHFAKDLRRHDVAPTDAVDVLKHELARLEV

VQTHFEFYEFFHEFMPREVRVKVVPDIVNKIPLPRHGVFSDILRGNNVQG

HHpred searches using the C-terminus of the B. mori and S. frugiperda CENP-T protein identify

the C-terminus of the G. gallus CENP-T with high probability 94.56 % and 94.2 % respectively

(Figure S2). HHpred searches using the full-length protein also identifies the G. gallus CENP-T

as a best hit though with lower probabilities (51.9 % for the B. mori and 50% for the S.

frugiperda protein). In this search, the alignment between the lepidopteran protein and G.

gallus CENP-T is restricted to their C-termini. A reciprocal HHpred search using the G. gallus

HFD helix 3 and CENP-T extension identified B. mori protein as best hit (Figure S2). Known

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vertebrate CENP-T proteins can also be identified as best hits after 2 jackhmmer iterations

using the B. mori CENP-T C-terminus (Figure S2). Additional arthropod CENP-T from the

whiteleg shrimp (Penaeus vannamei) and the pill woodlouse (Armadillidium vulgare) could be

predicted after 4 jackhmmer iterations. Finally, phyre2 predictions (Kelley et al., 2015) using

the full-length B. mori CENP-T protein predicts the known structure of the G. gallus CENP-T C-

terminus with high confidence (91.5%) while low sequence identity of the aligned regions

(14%). Iterative blastp and tblastn searches using lepidopteran CENP-T proteins against select

annotated insect proteomes, genome assemblies and assembled transcriptomes revealed

additional orthologs of CENP-T proteins in insects.

KWMTBOMO14835 and GSSPFG00001205001

BLASTP searches using both proteins against the nr database revealed only hits in Lepidoptera.

Iterative PSI-BLAST searches and jackhmmer searches using both proteins also revealed no

homologous hits in other insect orders. HHpred searches of GSSPFG00001205001 did not

reveal any hits with high probability. However, HHpred searches identified similarity between

the N-terminus of KWMTBOMO14835 and resolved structure of the S. cerevisiae Ctf19 protein

(CENP-P) (Hinshaw and Harrison, 2019) as best hit with high probability (93.58%) while only

13% amino acid identity. The aligned region also includes parts of the tandem RWD domains

from S. cerevisiae Ctf19. The N-terminus of KWMTBOMO14835 has predicted coil-coiled

regions (PCOILS, window 28). HHpred predictions of the trimmed protein without the coiled-

coil region reduced the probability of similarity to Ctf19 to 43.96%, which was also no longer

the best hit. We therefore refrain from assigning homology to Ctf19 without further functional

or structural validations and refer to the lepidopteran proteins as coiled-coil RWD-like

proteins.

KWMTBOMO09290 and GSSPFG00019785001

Both proteins contain N-terminal coiled-coil domains (PCOILS, window 28). HHpred searches

of both proteins without the N-terminal coiled-coil regions revealed similarities to RWD

domains of several kinetochore components including the resolved structure of the G. gallus

Spc24 globular domain (Nishino et al., 2013) and Mcm21 (CENP-O) subunit of the budding

yeast Ctf19 complex (Hinshaw and Harrison, 2019; Schmitzberger and Harrison, 2012) with

high and similar probabilities. Iterative jackhmmer searches of the trimmed B. mori protein

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without the N-terminal coiled-coil region identified hits in Hymenoptera in the second

iteration including Camponotus floridanus (E2AEP7, E value 0.0017). The third iteration

identified additional hits in the blattodean Zootermopsis nevadensis (A0A067RMY5, E value

0.00028), the arthropod Daphnia magna (A0A0P5Q3Q5, E value 6.5x10-7) and predicted

known vertebrate CENP-O proteins in Anabis testudineus (A0A3Q1H0S6, E value 0.0023 and

A0A3Q1H0T5, E value 0.0037). The fourth iteration identified the human CENP-O (E value

1.6x10-13). Given the similarity to multiple RWD containing kinetochore proteins and the lack

of experimental evidence we only refer to the lepidopteran proteins as coiled-coil RWD-like

proteins.

KWMTBOMO06154 and GSSPFG00011797001

HHpred searches did not identify high scoring hits for neither one of the two proteins.

GSSPFG00011797001 identified the recently resolved structure of the fungus Thielavia

terrestris CENP-H (Hu et al., 2019) but not as the best hit and with only 36 % probability. Still,

given the presence of CENP-I, CENP-M and the putative CENP-K, it will be worthwhile to test

if these proteins are part of a lepidopteran CENP-H-I-K-M complex. Hits in other insect orders

could not be predicted using psi-blast or jackhmmer.

LOC101741561 and GSSPFG00006732001

The B. mori homolog is not part of the most recent annotations (Kawamoto et al., 2019) , we

therefore refer to the ID of previous annotations present on NCBI Genebank in Figure 1.

GSSPFG00006732001 is likely misannotated because most other lepidopteran homologous

proteins including LOC101741561 only align to the C-terminal part of the protein. We derived

a new consensus sequence by aligning available TR2012b transcriptome data (Nègre et al.,

2006). The encoded ORF translates into the following protein:

>S. frugiperda CENP-K

MSSDRNKETRDAVKREIKEIQARCKHEWNIIDNSPLDAPNIDLEQINEKALCYIEGLGTGIQ

NSNTPITADDNLLTSQFLKEIRDKTGQVEEYTAFVRGSIHDIDAEINRLQTLIKITQEAKAR

PMLNKCEVQPEHVHRAKERFQVMKNELHSLIHSLFPNCDSLIIETMGQLMAEHLNEESNGYI

PVTAETFQIIELLKDMKIVTVNPYNKLEVKLSY

One of the EST that contain the full ORF is Sf2H05447-5-1 that is listed in Figure 1B. Iterative

PSI-BLAST searches using the B. mori protein revealed hits in Hymenoptera including Athalia

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rosae (LOC105689000, E value 5x10-5) after the 1st iteration. The 2nd iteration revealed hits in

Hemiptera including Bemisia tabaci (LOC109036758, E value 0.003), the Blattodean

Zootermopsis nevadensis (LOC110834781, E value 2x10-11) and the mollusk Mizuhopecten

yessoensis (centromere protein K-like, E value 0.001). The human CENP-K was identified after

the 3rd iteration. These analyses are consistent with previous predictions identifying CENP-K

in B. mori (Tromer, 2017). Jackhmmer searches of a N-terminal truncated version of the B.

mori protein without a coiled-coil region also revealed a hit in the phthirapteran Pediculus

humanus corporis after the 3rd iteration (E0VW71, E value 4.1x10-9).

KWMTBOMO11351 and GSSPFG00010096001

These are orthologs of Drosophila melanogaster bellwether, the alpha subunit F0F1 ATP

synthase subunit alpha recently been shown to interact with Cid during Drosophila

melanogaster male meiosis (Collins et al., 2018). Orthologs are present across insects and

diverse eukaryotes.

KWMTBOMO11557 and GSSPFG00019290001

HHpred searches aligns both proteins to the structure of the CHRAC 14 HFD (Hartlepp et al.,

2005) as best hits but low probabilities (Probability 49.3 % for KWMTBOMO11557 and 39.1%

for GSSPFG00019290001). Hits in other insect orders could not be predicted using psi-blast.

Jackhammer searches of both proteins converged after 2 iterations.

Sf2H06980-5-1 and KWMTBOMO014775

The S. frugiperda protein is not part of the S. frugiperda corn strain annotations but in the

annotations from the closely-related S. frugiperda rice strain (Gouin et al., 2017). We

therefore provide the ID from these annotations file. Both B. mori and S. frugiperda proteins

contain C-terminal coil-coiled regions. HHpred searches using only the N-terminal first 65

amino acids identifies the known structure of the human Nsl1 (Petrovic et al., 2016).Though

the human Nsl1 was not the best hit for neither one of the two lepidopteran proteins, given

the abundance of the protein in the Nnf1 and Dsn1 IPs we refer to it as Nsl1 candidate. These

data suggest that as in other eukaryotes, the lepidopteran Mis12 complex contains four

instead of only three components as recently described (Mon et al., 2017).

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Hemipteran CenH3

Hemipteran CenH3 fragments in Gerris buenoi genome assembly (Armisén et al., 2018) and

Aquarius paludum assembly (A. Khila, unpublished data) could be identified in TBLASTN

searches using D. melanogaster H3 as queries.

>Aquarius_paludum_Embryo_Assembly50_c84768)g2_il RRRSGVVALREIRHLQKSTNLLIPKLPFMRIVQEILQSYSTEPYRIQSRALEALQEMTEILM

VDLFSEAILCCIHAKRKTIMVQDMRLARRIRG

>Gerris_buenoi_ KZ651887.1 RRRSGVVALREIRHLQKSTNLLIPKLPFMRIVKEILQSYSTEPYRLQTQALEALQEMTEILM

VDLFSEAILCCLHAKRKTIMVQDMRLARRIRG

Plasmid construction

A list of plasmids generated in this study is provided in Table S3. For the kinetochore IPs, full-

length ORFs of S. frugiperda CENP-M, CENP-N, CENP-I, CENP-T, Dsn1 and Nnf1 fused to 3xFLAG

tags were cloned into pIBV5 (Invitrogen Cat#12550018) using the Gateway system (Invitrogen

Cat#11791019) from pENTR vectors (Invitrogen Cat#K240020). The genes encoding for S.

frugiperda CENP-T, CENP-I and CENP-N are not correctly annotated in the current assembly

(Gouin et al., 2017) as inferred by aligning the protein products to the B. mori homologs. We

inferred the correct ORF sequences from EST data and sequencing of the amplified PCR

fragments from Sf9 cDNA.

The following are the correct ORF sequences that were used for all experimental analyses:

>S. frugiperda CENP-I MADVDEIIDYIKSLKKGFDKDLFQNKIDELAYAVDTTGILYNDFHTLFKVWLNLSIPITKWV

SLGACLVPQNIVEDRTVEYALSWMLSNYEDQSTFSRIGFLLDWLTAAMECECIEMETLDMGY

DVFYVSLTYETLTPHAVKLVYTLTKPVDVTRRRVLELLDYARKREAKKNMFRQLXVLLGLFK

SYKPECVPEDIPAISIHAAFKKINPDLLARFKRNQENRNSVRRERHHLTWINPINSDRGRNK

KIDPLVPNVEFLNIGSKQYAEKEHQKNFLDFTDPVSVLQCSVQRSTSRPARIRALLCNVTGV

ALLAVASHTEQEFLSHDLHHLLNSCFLNISPHSYREKQDLLHRLAVLQHTLMQGIPVITRFL

AQYLPLWNERDYFAEILELVQWVSLDSPDHVTCVLEPLARAYHRAQPIEQCAILRSLNHMYC

NLVYASTRKRHHFMGTPPSPQVYALVLPKVATAISDMCDKELQVNPEQMVVLHSGVQGAAAR

ARGEARGGAAAGALAPRLALALPLLASSAALLDSVAELMILYKKIFTTAKQTNVITNTDTFE

KQMQVLEAYTSDLINCLYSEGALSDRNLGFVFSKLHPQLVEKLGSLMPDVDAKLSIRNSIAF

APYTYIQLDAIDHRDADNKLWFNAVIEQEFTNLSRFLKRAVTELRYQ

>S. frugiperda CENP-T MPKTKIPSPARPQGATTPKRKKSRGSRSVCSPALSTASGRLTSLIDQFKEKNMESFALSPLN

RRSILNDDTAIEEPRRQSWWKKLKEDSHEIMEVLEENKVADSGNNAIEELIDIEVLSQEKKE

93

YTLDLPESSDNESINSIVLPQRKLFTQKENKPQKKFGQFSDNRETLAKLHKTNTQGDKTVNV

GTRELFQNAKRSKPIFPAALLNISPNKTAMDKTKENILPEPKVRNIFGNRPANKRKNMFADF

VVSESEDEIPELQPRVFGFQKKLEQRRISSISKGREGSPASSITTDMEMDDWKLLPSSTMVE

NQLEDIVAGHTPKRARLSKLSEAKESEAGTLQTNTTGDKTKSSNKSIMSKNKSKSSPDAKDK

SLNSSRTTRSMLKNASKLEENTEPKQDTKTQSKISTKDNALDVSLSKATTKQNTSLNKSLRL

NKSRTRNSSKLNEEEMETDENVVKNAINDATRVISKNASSAAVASKSINEKERTITLKVHDK

ASETGSNKEEDDNNFVLQYEDEIVEEATDHNQINENKTTKIKNRNTIVIENDQETDVNESKS

NKSIQKEPKQAKTVEESVESQNEQSGNDNIDGNEIEENAIEQNHESHDGEKISRELNEESNN

SEGNDERNVTKDNKDQENDEEINESQENDEEINLSQESNRDEAVLSRVDEENESEVNEESEN

EDDVNESNESEEQNESQEVEAEVDESEEIEEQNESQEIENEANESQEVNEEADDSKVEENAS

EDEEENQEVENDEEENAEESQEVDNEESQEIENQEEFEVSQESEHEVSQEVENEEENEEEND

EGDQEVEQEEENQSEDDVEDQVEDQSQEIENDEENDEEENEESQEIENDEENVVESEAEVNE

SQEIEGDQEMDDSDEADRAIASDPEISDQDDNGADEMEVDDDDDNNEQESENEEETEGNQEE

SQEEQQEPSAEEIEESPNVTHDTTGRHRQKVLKSPEAILHDKTNQMDSFTAKGRNTSIRKTK

SMIKNLNIRPSLAPQRDSLAFSDGTRDSSAEGSGWDSHRTTRKTLRQTFGKDFTPRKSLRAL

VMEKSAKRQTEHMDLHSETSKYPQANSTELAEDSNGHVDDFVESDHEVSRRTRQTTLETYLQ

KIKQKNLENKVKMEELVRNSLKAPARDTLSLFKVPNKPAPRRLKPTQNKPRTQVKAITAFGE

LPTEVIEDMKYKPPKRFQPTNASWITKRLYKFLETKLEPKYDYKARVRAEKLVETIYHFAKD

LRRHDVAPTDAVDVLKHELARLEVVQTHFEFYEFFHEFMPREVRVKVVPDIVNKIPLPRHGV

FSDILRGNNVQG

>S. frugiperda CENP-N MPLEVFCVTALPEFGVRWRELSKAVRNARLPMQPASVARVLCRHVSKALTADEIEDIVARLR

LKLVATQPRTWHVIRLSEKTTEEPVTLTMRAVPGRITQALRKSKKTMRPEVQTVLLGDMLYL

SIQLVSEHKSGSALYVATPPGEPVALVSSVNMVGLIKATVEGLGYKSYEIADLHGRDIPSLL

RINDRAWNTNADHLAEIPEYAPTPIITETGIDYTYKAYDENYVENILGPNPPKITDLTIKTS

KSFFDRSRLDKNINITINIKTEDLAKSLKCWVSKGAIAPTSDLIKIFHQIKSNKISYTREDD

To express the C-terminus of S. frugiperda CENP-T a 166 amino acid C-terminal fragment that

contains the HFD and CENP-T extension was isolated to generate the S. frugiperda CENP-T-

HFDextension-FLAG pIBV5 expression clone.

For LacO/LacI tethering assays, pVS1-LacO (Addgene RRID: Addgene_33143 (Vodala et al.,

2008)) was modified to insert the blasticidine resistance cassette from pIBV5 into the BamH1

and Sac1 sites. To generate LacI-GFP pIBV5, the LacI-eGFP insert was amplified from

eGFP_N1_LacI pDEST (kind gift from Geneviève Almouzni lab, Nuclear Dynamics unit,

UMR3664, Institut Curie, Paris, France (Prendergast et al., 2016)) and cloned into pENTR and

pIBV5. To generate B. mori CENP-T-GFP-LacI pIZV5, the full-length coding sequence of B. mori

CENP-T from B. mori CENP-T-FLAG pIZV5, lacI from pCMV-lacI (kind gift from Geneviève

Almouzni lab, Agilent Cat#217450) and eGFP from eGFP_N1_LacI pDEST were isolated by PCR

and assembled into the shuttle vector pRS416 (kind gift from Heloise Muller, Nuclear

Dynamics unit, UMR3664, Institut Curie, Paris, France) using the yeast-based homologous

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recombination-based DNA assembly, described in detail in elsewhere (Gibson, 2011; Muller

et al., 2012). Briefly, S. cerevisiae BY4742 strains were transformed with the linear DNA

fragments and linearized pRS416, colonies were picked to isolate the resulting recombined

construct. The constructs were then transformed and amplified in E. coli. The S. frugiperda

CENP-T-GFP-LacI was isolated from this plasmid and cloned into pIZV5 (Invitrogen

Cat#V800001) using KpnI and XbaI. To generate the B. mori N-CENP-T(201-1013)-GFP-LacI

pIZV5, a truncated B. mori CENP-T-GFP-LacI fragment (without the part coding for the first 200

amino acids was isolated PCR and cloned into pIZV5 using BamHI and XhoI sites.

To generate the B. mori CENP-T-FLAG pIZV5 construct, the coding sequence of B. mori CENP-

T was isolated from a BmN4 cDNA library, fused to the 3xFLAG tag by PCR amplification and

cloned into pIZV5 using BamHI and XhoI. To generate the B. mori CENP-T RNAi-resistant clone

(B. mori CENP-Tres-FLAG pIZV5), we ordered a Gblocks gene fragment (IDT) to recode an

internal sequence of B. mori CENP-T from amino acid 221 – 334. A 5’ fragment of B. mori CENP-

T that contains the recoded part was then assembled with the 3’ fragment of B. mori CENP-T

fused to a 3xFLAG into pRS416 using yeast-based homologous recombination and

subsequently cloned into the into the BamHI and XhoI sites of pIZV5. To generate the B. mori

N-CENP-Tres-FLAG pIZV5 and B. mori C-CENP-Tres-FLAG pIZV5, B. mori CENP-Tres-FLAG

pIZV5 was used as a template to isolate 5’ or 3’ truncated fragments to clone those into pIZV5

using BamHI and XhoI. The constructs expressed CENP-T without the first 200 or last 112

amino acids, respectively.

To generate protein antigens for antibody production, we purified protein fragment expressed

in bacteria. To generate the S. frugiperda SUMO-6xHis-CENP-T (1-221) pT7 and B. mori SUMO-

6xHis-CENP-T (1-218) pT7 bacterial expression constructs the N-terminal domains of S.

frugiperda (1-221aa) and B. mori (1-218aa) CENP-T were cloned into the pT7-His-SUMO vector

(gift from Ahmed El-Marjou, recombinant protein platform, Institut Curie) which included N-

terminal 6X His and SUMO tags using Gibson Assembly (Gibson et al., 2009).

To generate the B. mori 6xHis-Spc24(73-162)-Spc25(70-211) pRSF-DUET1(Sigma Cat#71341)

bacterial expression constructs, the globular domain of B. mori Spc24 (73-162aa) was cloned

with an N-terminal 6X His-tag into the first cassette of the pRSF-Duet1 vector using the Bam

95

HI and PstI restriction sites. For dual expression of Spc24 and Spc25, the globular domain of B.

mori Spc25 (70-211aa) was also cloned into the second cassette of the same pRSF-Duet1

vector using the Nde I and Xho I restriction sites. To generate the B. mori 6xHis-Spc24(73-

162)-Spc25(70-211) pFASTBac-Dual for the recombinant baculovirus generation to evaluate

the Spc24/25 antibody, the Spc24 and Spc25 fragments were cloned into the BamHI and PstI,

and KpnI and XhoI sites of pFASTBac-Dual, respectively. To generate the B. mori 6xHis Dsn1(1-

100) pRSF-DUET1 and S. frugiperda 6xHis Dsn1(1-99) pRSF-DUET1, N-terminal region of B.

mori (1-100aa) and S. frugiperda (1-99aa) Dsn1 cloning into pRSF-Duet1 with N-terminal 6X

His-tag BamHI + PstI. All plasmids generated are listed in Table S3.

Construction of cell lines

Around 1-5 g of plasmid DNA was transfected into 106 BmN4 or Sf-9 cells using Cellfectin II

(Gibco Cat#10362100) according to the manufactor’s instructions. For IF experiments cells

were grown on coverslips before transfections. For the generation of stable polyclonal cell

lines, antibiotics were added 48 hours after transfection (300 g/ml Zeocin (Gibco

Cat#R25001) or 40 g/ml Blasticidin (Gibco Cat#R21001)). Selection was continued until no

viable untransfected cells were observed.

Protein expression and affinity purification

The E. coli Bl21DE30 plys pRare (Sigma Cat#71400) was transformed with bacterial expression

vectors containing the recombinant genes. Bacterial cultures (4 L) were initially grown at 37

°C, 220 rpm and then induced with 0.5 mM IPTG (Euromedex Cat#EU0008-A). Following

expression at 37 °C for 4 h, cultures were centrifuged at 6000 xg for 15 min to pellet the cells.

Cell pellets were resuspended in Wash Buffer (20 mM Tris pH 8, 300 mM NaCl, 10 mM MgCl2,

25 mM imidazole). The resuspended pellets were incubated at 4 °C on a roller with the

addition of one tablet cOmplete Protease Inhibitor Cocktail (Roche Cat#11697498001), Triton-

X-100 (1% final concentration), and lysozyme (1 mg/mL final concentration). The mixtures

were sonicated at 30% amplitude for 8 min total (30 sec pulses) in a Branson Digital Sonifier

SFX550 (Branson Ultrasonics Corp.). Cell lysates were centrifuged at 30 000 xg for 1 h at 4 °C.

The soluble fraction was removed and passed through 0.45 μm filter units. The lysates were

loaded onto Protino Ni-TED 1000 columns (Machery-Nagel Cat#745110.50) that were pre-

equilibrated with Wash Buffer. The columns were washed with 2-3 column volumes of Wash

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Buffer and proteins were eluted with the following buffer (20 mM Tris, 250 mM imidazole,

300 mM NaCl, pH 8.0). The SUMO tags were cleaved from the CENP-T proteins by digesting

the eluates at 4 °C with SUMO-Protease (enzyme produced by Ahmed El Marjou, recombinant

protein facility, Institut Curie) (final concentration 0.6 μg/mL). The eluates were loaded and

migrated in Bolt 4-12% Bis-Tris Plus denaturing gels (Invitrogen Cat#NW04120BOX). The

correct-sized bands corresponding to the proteins without the SUMO tag were excised from

the gels and sent to Covalab (Villeurbanne, FR) for generation of antibodies in rabbits. For the

generation of antibodies against CENP-T and Dsn1, a 1:1 mix of B. mori and S. frugiperda CENP-

T or Dsn1 protein fragments were injected into rabbits. For the generation of the antibody

recognizing Spc24 and Spc25 proteins, the isolated B. mori Spc24 and Spc25 protein fragments

were injected.

Validation of CENP-T and Dsn1 antibody specificity

For each IP experiment, one confluent flask of BmN4 cells was used. Immunoprecipitation was

performed as described previously (Skene and Henikoff, 2015) omitting the cross-linking step

and with some modifications. Briefly, cells were spun down and washed with PBS with

cOmplete Protease Inhibitor Cocktail (Roche Cat#11697498001). Cells were resuspended in

140 μl lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl (pH 8.1)) with protease inhibitors

and incubated for 10 minutes at 4 °C. 1350 l IP dilution buffer (1% Triton X-100, 2 mM EDTA,

150 mM NaCl, 20 mM Tris-HCl (pH 8.1)) with protease inhibitors and 4.5 μl CaCl2 (1 M) were

added and samples were incubated for 2 minutes at 37 °C. Chromatin was digested using I

UNIT of MNase (Sigma Cat#N3755-500UN) for 15 minutes at 37 °C. The reaction was stopped

by adding 30 μl EDTA and 60 μl EGTA followed by mild shearing and solubilization step using

the Covaris E220 Evolution ultrasonicator (Covaris) (150 sec, peak power 75, duty factor 10,

cycles/burst 200). Samples were spun down for 5 minutes at 16000 xg and the soluble extract

was added to 50 l magnetic Dynabeads Protein A (Invitrogen Cat#10002D) covalently

conjugated to 5 l of rabbit polyclonal anti-CENP-T (this paper) or anti-Dsn1 (this paper)

immunoserum or 10 g anti-FLAG M2 antibody (Sigma Cat#F1804; RRID: AB_262044) as a

control. Immunoprecipitation was performed for 15 minutes at room temperature and

samples were washed three times in PBS. Samples were digested on beads for MS analyses

(Figure S4A and B) (see below).

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Validation of B. mori Spc24/25 antibody specificity

The 6xHis-Spc24(73-162)-Spc25(70-211) pFASTBac-Dual construct was used to generate

recombinant baculovirus DNA. Briefly, pFASTBac-Dual constructs were transformed into

DH10bacLL strain to produce and isolate the recombinant baculovirus backbone.

Recombinant baculoviruses were amplified in Sf9 cells to the generate high-titer virus stocks

of B. mori Spc24/Spc25 baculovirus. 500 µl of the recombinant Spc24/Spc25 expressing

baculovirus stock (MOI 100) were used to infect 50ml Sf9 cells (106 cells/ml) for 3 days. To

obtain total cell extracts, the cell pellet was resuspended in buffer A (20 mM Tris pH 8, 300

mM NaCl, 5% glycerol, one tablet cOmplete Protease Inhibitor Cocktail (Roche

Cat#11697498001) and 20µl DNase I (Roche Cat#04716728001)) and incubated for 20 min at

4°C. The samples were sonicated at 20% amplitude for 3 min and 30 sec total (30 sec pulses)

in a Branson Digital Sonifier SFX550 (Branson Ultrasonics Corp.) and centrifugated for 30min

at 8000 rpm, at 4°C. The supernatant was used for the subsequent experiments. The lysates

were loaded onto Protino Ni-TED 1000 columns (Machery-Nagel) that were pre-equilibrated

with Wash Buffer. After a 30 min incubation at 4°C, the columns were washed in Wash Buffer

with 20 mM imidazole followed by several elution steps with increasing concentrations of

imidazole (50 mM, 100 mM, 200 mM and 500 mM). Proteins were separated on 4-20% Tris

glycine gels (Invitrogen Cat#XP04200BOX) and visualized using InstantBlue™ (Sigma

Cat#ISB1L).

For protein blot analysis, samples were separated on Bolt 4-12% Bis-Tris Plus gels (Invitrogen

Cat#NW04120BOX) and transferred to a PVDF membrane (Bio-Rad Cat#170-4272) using the

Trans-Blot Turbo Transfer System (1.3 A, 25 V, 10min). The membrane was blocked using the

Odyssey Blocking buffer (LI-COR Cat#927-50000) before primary antibody incubation (rabbit

polyclonal anti-Spc24/Spc25 (this paper), 1:1000 dilution and mouse monoclonal anti-6xHis,

1:1000 dilution (Sigma Cat#ab18184; RRID:AB_444306)) and secondary antibody incubation

(IRDye 680RD Goat anti-Rabbit IgG (LI-COR Cat#926-68071; RRID:AB_10956166), IRDye

800CW donkey anti-mouse IgG (LI-COR Cat#926-32212; RRID:AB_621847), dilution 1:10000).

The signals were visualized on an Odyssey LI-COR scanner (Figure S4C).

Affinity co-immunoprecipitations

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Cultures of the following Sf9 strains expressing full length or partial S. frugiperda kinetochore

proteins fused to a C-terminal or N-terminal 3XFLAG tags or control wild-type Sf9 cells were

grown to exponential phase in Sf-900 II SFM medium (Gibco Cat#10902-088): CENP-I, CENP-

M, CENP-N, CENP-T, CENP-T-HFDextension, Dsn I and Nnf1. For each strain, 3 x 109 cells were

harvested by centrifuging for 10 min at 300 xg, and the pellets were washed twice in cold PBS.

The pellets were resuspended in 5 mL HDG150 Buffer (20 mM HEPES pH 7.0, 150 mM KCl, 10%

glycerol, 0.5 mM DTT, 1 tablet cOmplete Protease Inhibitor), and then cells were disrupted

with 50 strokes in a dounce homogenizer at 4 °C. The dounced fraction was centrifuged at

1700 xg for 10 min at 4 °C, and the nuclei (lower fraction) were gently resuspended with 5 mL

HDG150 Buffer and re-centrifuged in the same conditions. The nuclei were resuspended in a

final volume of 5 mL using HDG150 Buffer. Nuclear extracts were prepared by passing the

nuclear fraction 10 times through a 20G 1 ½” needle (0.9 x 38 mm) and then 5 times through

a 25G 3/8” needle (0.5 x 16 mm). The nuclear extracts were centrifuged at 20 000 xg for 10

min at 4 °C in microcentrifuge tubes. The pellets were resuspended in HDG150 Buffer and

pooled together at a final volume of 5 mL. To prepare the chromatin, the nuclear extracts

were digested with ~40 units MNase (Sigma Cat#N3755-500UN) for 1 hour at 4 °C on a roller,

3 mM CaCl2 was added to the digestions. The MNase digestions were stopped by adding 250

µL of 0.2 M EGTA. To solubilize the digested chromatin, 10 mL of HDG400 Buffer (20 mM

HEPES pH 7.0, 400 mM KCl, 10% glycerol, 1 mM DTT, 0.05% NP-40, 1 tablet cOmplete Protease

Inhibitor) was added to the samples and incubated for 2 h at 4 °C on a roller. The samples

were centrifuged at 8000 xg for 10 min at 4 °C. The supernatants were saved to bind to the

anti-FLAG M2 beads (Sigma Cat#M8823; RRID:AB_2637089). The M2 magnetic beads were

prepared according to the manufacturer’s recommendations. The digested chromatin

samples were incubated with 150 µL anti-FLAG M2 beads. The beads were washed four times

with 1 mL HDGN320 Buffer (20 mM HEPES pH 7.0, 320 mM KCl, 10% glycerol, 1 mM DTT,

0.05% NP-40, 1 tablet cOmplete Protease Inhibitor). For proteomic analyses of full-length

kinetochore protein IPs, beads were boiled in sample buffer to first run those into gels (see

below). For proteomic analyses of the CENP-T-HFDextension IP, proteins were directly

digested on beads (see below). For the silver stainings of Sf9 kinetochore IP samples shown in

Figure S1, M2 beads were incubated with FLAG peptide (Sigma Cat#F4799) diluted to a final

concentration of 150 μg/mL in 750 µL TBS Buffer (50 mM Tris-HCl, pH 7.4, with 150 mM NaCl)

for 1 h at 4 °C on a roller. The supernatants were removed and the eluates were concentrated

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in Amicon Ultra-0.5 mL 3K MWCO filters (Sigma Cat#UFC5003) according to the

manufacturer’s recommendations. Samples were loaded and migrated on Novex 16% Tris

Glycine Precast Gels (Invitrogen Cat#XP00162BOX). Silver stains were performed on the gels

using the Pierce Silver Stain Kit (Thermo Fisher Scientific Cat#24612) according to the

manufacturer’s instructions. Several bands were excised for mass spectrometry.

Proteomics and Mass Spectrometry Analysis

IP enriched proteins of full-length kinetochore protein IPs and control samples were separated

on 10% NuPAGE 10% Bis-Tris protein gel (Invitrogen Cat#NP0301BOX) and stained with

colloidal blue (LabSafe GEL Blue GBiosciences Cat#786-35). SDS-PAGE was used with short

separation as a clean-up step, and 4 gel slices were excised. Gel slices were washed and

proteins reduced with 10 mM DTT (Euromedex, Cat#EU0006-B) prior to alkylation with 55 mM

iodoacetamide (Sigma Cat#I6125). After washing and shrinking the gel pieces with 100%

MeCN (Merck Cat#1.00099), in-gel digestion was performed using trypsin/Lys-C (Promega

Cat#V5071) overnight in 25 mM NH4HCO3 (Fluka, Cat#09830) at 30 °C. Peptides were then

extracted using 60/35/5 MeCN/H2O/HCOOH (Fluka Cat#94318) and vacuum concentrated to

dryness. Protein on beads samples (CENP-T-HFDextension IP and CENP-T and Dsn1 antibody

validations) were washed twice with 100 µL of 25 mM NH4HCO3 and submitted to on-beads

digestion with 0.2 µg of trypsin/Lyc-C for 1h. Digested sample were then loaded onto

homemade C18 StageTips for desalting and peptides were eluted using 40/60 MeCN/H2O +

0.1% formic acid and vacuum concentrated to dryness. Gel samples were chromatographically

separated using an RSLCnano system (UltiMate 3000 RSLCnano, Thermo Fisher Scientific)

coupled to an Orbitrap Fusion mass spectrometer (Q-OT-qIT, Thermo Fisher Scientific with a

Nanospay Flex ion source (Thermo Fisher Scientific) and bead samples were also analyzed with

a Q Exactive HF-X mass spectrometer. Peptides were first trapped on a C18 precolumn (300

µm inner diameter x 5 mm; Invitrogen Cat#160454) at 20 µl/min or 2.5 µL/min with buffer A

(2/98 MeCN/H2O in 0.1% formic acid). After 3 min or 4 min of desalting, the precolumn was

switched on line with the analytical C18 column (75 µm inner diameter x 50 cm; nanoViper

Acclaim PepMapTM RSLC, 2 μm, 100Å, Thermo Fisher Scientific Cat#164535) equilibrated in

buffer A. Separation was then performed with a linear gradient of 5% to 25% or 30% buffer B

(100% MeCN in 0.1% formic acid) at a flow rate of 300 nL/min over 100 min or 91 min. MS full

scans were performed in the ultrahigh-field Orbitrap mass analyzer in ranges m/z 400–1500

100

or m/z 375-1500 with a resolution of 120 000 at m/z 200, ions from each full scan were HCD

fragmented and analyzed in the linear ion trap or orbitrap. For identification, the data were

merged and searched against the S. frugiperda corn strain proteome (Gouin et al., 2017) using

SequestHF through Proteome Discoverer (version 2.2, Thermo Fisher Scientific) with the S.

frugiperda CENP-T, CENP-I, CENP-N, Nsl1 candidate and Spc25 (which were all not correctly

annotated in the S. frugiperda corn strain assembly) manually added to the proteome

database. For identification of proteins in the B. mori IPs, the data were searched against the

B. mori proteome. Enzyme specificity was set to trypsin and a maximum of two-missed

cleavage sites were allowed. Oxidized methionine, Carbamidomethyl cysteines and N-

terminal acetylation were set as variable modifications. Maximum allowed mass deviation was

set to 10 ppm for monoisotopic precursor ions and 0.6 Da for MS/MS peaks. The resulting files

were further processed using myProMS (Poullet et al., 2007) v3.6. FDR calculation used

Percolator and was set to 1% at the peptide level for the whole study. For the kinetochore IPs

on full-length lepidopteran proteins identified proteins that were at least four-fold enriched

over the control (the two controls were combined) with a minimum of seven derived peptides

were analyzed further. For the S. frugiperda CENP-T-HFD-FLAG IP (Figure S1B) proteins that

were at least four-fold enriched over the control with at least 5 derived peptides were selected

for further analyses (see above). For Figure 1, known kinetochore homologs or proteins with

at least seven derived peptides and that were enriched at least four-fold in three kinetochore

immunoprecipitates are listed.

Immunofluorescence

Cells were grown on glass coverslips and fixed with ice cold MeOH (anti-CENP-T and anti-

Spc24/25), ice cold acetone (anti-Dsn1) and 4% PFA (anti-tubulin), followed by

permeabilization using 0.3% Triton X-100 in PBS and blocked in 3% BSA-PBS. The following

antibodies were used: rabbit polyclonal anti-CENP-T (this paper, rabbit 045), rabbit polyclonal

anti-Spc24/25 (this paper, rabbit 1621016) and polyclonal rabbit anti-Dsn1 (this paper, rabbit

1615031) generated by Covalab (Villeurbanne, FR) at the dilution 1:1000, anti-α-tubulin

monoclonal Alexa Fluor 488 (Thermo Fisher Scientific Cat#53-4502-80; RRID:AB_1210526) at

1:1000, anti-FLAG M2 mouse monoclonal (Sigma Cat#F1804; RRID:AB_262044) at 1:1000,

anti-phospho Histone H3-Ser10 rat monoclonal (Sigma Cat#MABE939) at 1:1000. For

fluorescent conjugated secondary antibodies, we used goat anti-rabbit IgG Alexa Fluor 568

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(Thermo Fisher Scientific Cat#A-11011; RRID:AB_143157) at 1:1000, goat anti-rat IgG Alexa

Fluor 568 (Thermo Fisher Scientific Cat#A-11077; RRID:AB_2534121) at 1:1000, goat anti-rat

IgG Alexa Fluor 488 (Thermo Fisher Scientific Cat#A-11006; RRID:AB_2534074) at 1:1000, goat

anti-mouse IgG Alexa Fluor 488 (Thermo Fisher Scientific Cat#A-11029; RRID:AB_2534088) at

1:1000, goat anti-mouse IgG Alexa Fluor 568 (Thermo Fisher Scientific Cat#A-11004;

RRID:AB_2534072) at 1:1000 and goat anti-rat IgG Alexa Fluor 633 (Thermo Fisher Scientific

Cat#A-21094; RRID:AB_2535749) at 1:1000. DNA was stained with DAPI (Sigma Cat#D9542)

and samples were mounted in Vectashield Antifade Mounting Medium (Vector Laboratories

Cat# H-1000; RRID:AB_2336789).

For anti-tubulin staining cells were fixed three and five days after RNAi-mediated depletion

using a protocol for the preservation of the whole cytoskelon (Bosch Grau et al., 2013). Cells

were washed with PBS for 5 minutes, then incubated for 10 min at room temperature in 1 mM

dithiobis(succinimidyl propionate, DSP) (Thermo Fisher Scientific Cat#22585) in Hank’s

balanced salt solution (HBSS) (Gibco Cat#14025050), followed by an incubation for 10 min at

room temperature in 1 mM DSP in microtubule-stabilizing buffer (MTSB). Cells were next

washed for 5 min in 0.5% Triton X-100 in MTSB and then fixed in 4% PFA in MTSB for 15 min

at room temperature. After a 5 min wash in PBS, cells were incubated for 5 min in 100 mM

glycine in PBS, then washed again in PBS for 5 minutes and finally nuclei were stained with

DAPI (Sigma Cat#D9542) and samples were mounted in Vectashield Antifade Mounting

Medium (Vector Laboratories Cat# H-1000; RRID:AB_2336789).

Microscopy

Images were acquired on Zeiss Axiovert Z1 light microscope. Z-sections were acquired at

0.2 m steps using 100X 1.4 NA oil objective.

Quantification of fluorescence intensity was performed using the Fiji software (Schindelin et

al., 2012) on unprocessed TIFF images. Mitotic cells (H3S10ph positive) were quantified. For

RNAi depleted cells, we first annotated the cells using an automated system (kind gift from

Solène Hervé, Fachinetti lab, UMR144, Institut Curie, Paris, France). H3S10ph signals were

then used as markers to manually select, using the freehand selections tool, the nuclear area.

The mean fluorescence intensity of each nucleus was measured and corrected for background.

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For background correction, the average of mean intensities of three random circular regions

of fixed size (30x30 pixels) placed outside nuclear areas was determined.

To quantify the fluorescent signal intensities for LacO/LacI tethering assays, the mean

fluorescence intensities of CENP-T, Dsn1 and Spc24/25 signals were first measured in circular

regions of fixed size (10x10 pixels) overlapping GFP-LacI fusion protein foci (visualized by the

GFP signals). Then, the mean fluorescence intensity of CENP-T, Dsn1 and Spc24/25 at

endogenous loci was determined as the average in three random circular regions of fixed size

(10x10 pixels) placed over the mitotic chromosomes. As before, the background intensity was

also measured as the average of mean fluorescence intensities of three random circular

regions of fixed size (10x10 pixels). Both the mean fluorescence intensities overlapping the

GFP foci or the endogenous loci were corrected with this background value. For the

quantifications in Figure 4, the ratio between the mean fluorescence intensity over the GFP

foci and over endogenous loci was calculated and plotted. A ratio above 1 means that the

respective kinetochore components are enriched over GFP fusion protein foci and therefore

indicates recruitment by the tethered protein. In contrast, a ratio around 1 or below indicate

that kinetochore components are not preferentially enriched or are even depleted over the

GFP fusion protein foci compared to endogenous loci. While the corrected values of the

fluorescence intensities at the endogenous loci were always positive, the corrected

fluorescence intensities overlapping the GFP foci can be negative in cases where the

immunosignal was low and even below the average background intensity. Therefore, the ratio

of (corrected immunosignal overlapping the GFP foci/ corrected intensities at the endogenous

loci) can be negative.

For statistical analysis the Mann-Whitney test (unpaired, non-parametric test) was used to

compare two ranks, using GraphPad Prism version 8.12 for Mac (GraphPad Software,

https://www.graphpad.com/scientific-software/prism/). Differences were considered

statistically significant at values of P values <0.05.

FRAP

FRAP experiments were performed on a spinning-disk microscope (inverted Eclipse Ti-E

(Nikon) and Spinning-disk CSU-W1 (Yokogawa) integrated in Metamorph software by Gataca

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Systems) using a CFI SR Plan Apochromat IR 1.27 NA 60x water objective, the 488-nm laser

line for GFP (Gataca Systems) and the FRAP module ILAS2 (Gataca Systems). 1 or 5 images

were taken before the bleach pulse. The bleach pulse was performed on a circular area of

fixed size (20x20). After bleaching, 50-70 pictures were acquired. Quantitation of relative

fluorescence intensities was done according to Phair and Misteli (Phair and Misteli, 2000) and

with the help of Christophe Klein (CICC, UMRS1138 Centre de Recherche des Cordeliers de

Jussieu) using Fiji and Excel (Microsoft) software. Due to the fast recovery observed and low

fluorescence intensity of the GFP-tagged proteins, we could not determine the recovery half-

times and residence times.

RNAi-mediated knock-down

BmN4-SID1 cells were grown on coverslips and incubated with 400pg/l dsRNA for three days.

After three days, the medium was change to add another 400pg/l dsRNA. After three or five

days, cells were fixed and processed for IF as described. Primers used to generate the DNA

templates fused to T7 promoter for dsRNA generation are listed in Table S4.

RNA blot analyses

Total RNA was isolated using TRIzol (Invitrogen Cat#15596018) following the manufactor’s

instruction. RNA blots were performed using around 10 μg total RNA (CENP-T, CENP-I, CENP-

N, Nsl1, Mis12, Spc25) or polyA-selected mRNAs from 20 μg total RNA (CENP-M, Spc24) per

lane. RNA samples from cells incubated for five days with respective dsRNAs were treated

with glyoxal using NorthernMax-Gly Sample Loading Dye (Thermo Fisher Scientific

Cat#AM8551). The samples were loaded on a 1% agarose gel prepared using NorthernMax-

Gly Gel Running Buffer (Thermo Fisher Scientific Cat#AM8678) according to the

manufacturer’s instructions. The RNA was blotted onto a Nytran membrane in 20X SSC (175.3

g NaCl, 88.2 g sodium citrate in 1.0 l water adjusted to pH 7) using the TurboBlotter System

(Bio-Rad Cat#1704155). After UV crosslinking RNA to the membrane, glyoxal treatment was

reversed by incubating the membrane in 10 mM Tris-HCl pH 8 for 20 minutes at room

temperature. The membrane was incubated in 12 ml QuickHyb solution (Agilent Cat#201220)

with 1.0 mg salmon-sperm ssDNA (Sigma Cat#31149) for 1 hour at 65°C. Body-labeled

antisense riboprobes against kinetochore mRNAs and the loading control were prepared by

using PCR products as templates for in vitro transcription (MAXIscript T7 Transcription kit,

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Thermo Fisher Scientific Cat#AM1312). A radiolabeled probe against B. mori Rpl32 mRNA

served as loading control. After an overnight hybridization at 65°C with radio-labelled probes,

the membrane was washed twice in 2X SSC, 0.1% SDS for 5minutes and once in 0.2X SSC, 0.1%

SDS for 30 minutes. The membranes were exposed to phosphoimaging plates and analyzed

using a Typhoon TRIO Imager.

CRISPR-mediated genome editing in B. mori

The non-diapause strain N4 maintained at the University of Tokyo was used. All larvae were

fed with fresh mulberry leaves or artificial diet SilkMate (NOSAN SilkMate PS) under a

continuous cycle of 12-h light and 12-h darkness at 25°C. Unique single-guide RNA (sgRNA)

target sequences in the silkworm genome were selected using CRISPRdirect

(https://crispr.dbcls.jp/) (Naito et al., 2015). The sequence specificity in N4 strain was also

checked by SilkBase (http://silkbase.ab.a.u-tokyo.ac.jp) (Kawamoto et al., 2019) . Primers

used for sgRNA transcription in vitro are listed in Table S4. The sgRNA was transcribed in vitro

according to a method reported previously (Bassett et al., 2013). A mixture of sgRNA (400

ng/µL) and Cas9 Nuclease protein NLS (120 ng/µL; NIPPON GENE Cat#319-08641) in injection

buffer (100 mM KOAc, 2 mM Mg(OAc) 2, 30 mM HEPES-KOH; pH 7.4) was injected into each

egg within 3 h after oviposition (Yamaguchi et al., 2011). The injected embryos were incubated

at 25°C in a humidified Petri dish until hatching. Injected individuals were crossed with non-

injected individuals to obtain G1 broods. To identify G1 moths in which mutant alleles were

transmitted from G0, genomic DNA was extracted from a G1 adult leg using the hot sodium

hydroxide and Tris (HotSHOT) method (Truett et al., 2000). Genomic PCR was performed using

KOD OneTM (TOYOBO Cat# KMM-101) under the following conditions: 35 cycles of

denaturation at 98°C for 10 s, annealing at 60°C for 5 s, extension at 68°C for 5 s. Primers used

for mutation screening are listed in Table S4. The PCR products were denatured and

reannealed at 95°C for 10 min, followed by gradual cooling to 25°C. Mutations at the target

site were detected by heteroduplex mobility assay using the MultiNA microchip

electrophoresis system (SHIMADZU) with the DNA-500 reagent kit (SHIMADZU Cat#292-

27910-91) (Ansai et al., 2014; Ota et al., 2013). A mutation at the target site was sequenced

using BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems Cat#4337454) and

ABI PRISM® 3130xl Genetic Analyzer (Applied Biosystems). We maintained the mutant line

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and obtained eggs carrying the homozygous mutation by crossing between two heterozygous

mutants.

QUANTIFICATION AND STATISTICAL ANALYSIS

Statistical details of experiments are detailed in the Figure legends, including the number of

biological replicates (n= number of cells), the definition of center, dispersion and precision

measures (mean, SEM and SD). Statistical analyses were performed with GraphPad Prism 8.12

for Mac.

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107

Supplementary Figures

108

109

Figure S1. Composition of CenH3-deficient kinetochore in S. frugiperda. A) Silver staining of SDS-PAGE separating protein

complexes identified by immunoprecipitation using anti-FLAG affinity steps followed by elutions using the FLAG peptide from

Sf9 cell lines stably expressing 3xFLAG-tagged inner kinetochore components (CENP-M, CENP-I, CENP-N and CENP-T) and outer

kinetochore components (Dsn1 and Nnf1). Wild-type Sf9 cells (Control) were also used for affinity purifications with anti-FLAG

antibodies. Kinetochore proteins used as baits are highlighted in red. The identity of several individual bands was determined

by mass spectrometry (MS) analysis of digested peptides (blue). The theoretical size of other components identified in the IPs

with at least 5 matching peptides are indicated. B) Absence of detectable lepidopteran CENP-W candidates in S. frugiperda

FLAG-tagged CENP-T HFD and 2-helix extension (CENP-T-HFDextension-FLAG) immunoprecipitates. Top: Table lists the

number of peptides and coverages of S. frugiperda proteins identified by mass spectrometry enriched in the CENP-T-

HFDextension-FLAG IP over the control. For MS analyses, samples were directly digested on beads. The corresponding

homologs in B. mori and descriptions based on homology predictions are listed alongside. Two small proteins of unknown

function are highlighted in green. Bottom: Depletion of B. mori homologs of the two small S. frugiperda proteins found in

CENP-T-HFDextension-FLAG IPs had no detectable mitotic and CENP-T recruitment defect. Graph showing the percentage of

mitotic cells (H3S10ph positive cells) three days after RNAi-mediated depletions of corresponding mRNAs (n= number of cells,

± standard error of the mean). Representative images of mitotic BmN4-SID1 cells showing the levels of endogenous CENP-T in

cells upon depletion of two small B. mori proteins. Scale bar: 10μm. While we identified two small proteins with sizes similar to known CENP-W homologs in IP experiments pulling down S. frugiperda CENP-T C-terminus, RNAi-mediated depletions of B.

mori homologs do not increase the number of mitotic cells or abolish CENP-T localization to mitotic chromosomes. Both

potential protein products (LOC105842400 is annotated as a non-coding RNA) have no detectable similarity to known CENP-

W homologs.

A)

B)

110

A) B)

C)

111

Figure S2. Prediction and evolution of insect CENP-T homologs. A) and B) Prediction of known CENP-T homologs using the C-

terminus of the B. mori CENP-T protein. Homology searches (arrows) and corresponding E values that link the C-terminus of

the B. mori CENP-T protein to other known CENP-T homologs including Calypte anna (Aves), the human CENP-T and the G.

gallus CENP-T domain structure indicated by species name and UniProt or PDB IDs. While the first jackhammer iteration only

picked up insect homologs, the best hits of the second iteration are known CENP-T homologs. The best hit of the HHpred search

within the PDB is the G. gallus CENP-T. The reciprocal best hit of a search using the G. gallus CENP-T HFD helix 3 and extension

(amino acid 639-689) against the B. mori proteome was the B. mori CENP-T. B) Multiple alignment of the C-terminus of

vertebrate and insect CENP-T proteins. B. mori (above) and G. gallus (below) secondary-structure predictions are derived from

the Jpred4 (α-helical regions are in red; β-strands in yellow). Background coloring of the residues is based on the clustalX

coloring scheme. C) Phylogenic distribution of known and insect CENP-T proteins as well as various additional histone fold

proteins. Maximum-likelihood phylogeny of HFD with known CENP-T proteins in orange and newly identified insect CENP-T

proteins in red. Bootstrap values above 80 are indicated by purple circles on the branches. The scale bar measures the

evolutionary distance in amino acid substitutions per site. The phylogenetic relationship between the insect proteins (red) and

other CENP-T proteins (orange) cannot be unambiguously resolved. Nevertheless, we refer to the insect proteins as CENP-T

because of the common sequence architecture with other CENP-T proteins; remote homology predictions identifying known

CENP-T proteins as best hits outside of insects (Figure S2A and B) and the experimental evidence of kinetochore participation

and outer kinetochore recruitment. Notably, the branch at the root of the insect CENP-T proteins clade indicates a high degree

of protein sequence divergence of the insect homologs, which could explain why their putative homology to other CENP-T

homologs has been missed in previous searches.

112

A)

B) C)

D)

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Figure S3. Depletion of kinetochore components affects mitotic progression in B. mori cells. A) RNA blot analyses showing

mRNA levels encoding for various kinetochore components in control (CTRL - RNAi against GFP) and depleted cells (KD). The

star indicates the band of the respective mRNAs. A probe against Rpl32 mRNAs were used as loading control. The size marker

is in base pairs. An RNA probe targeting the Dsn1 transcript did not reveal any signal and is therefore not included in this

figure. The efficient depletion of Dsn1 protein is confirmed in our IF data analyses (Figure 3). We were unable to assign one

band to the CENP-M transcript indicating the possibility of multiple expression isoforms. The signal of all bands was decreased

upon RNAi treatment indicating efficient depletion of CENP-M transcript levels. B) Quantification of the percentage of mitotic

E) F)

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cells (H3S10ph positive cells) five days after RNAi-mediated depletion of various kinetochore components (n= number of cells,

± standard error of the mean). C) Representative images of mitotic cells stained with anti-tubulin used to classify mitotic

defects observed at three days after depletion of outer kinetochore proteins Mis12, Nsl1 and Spc25. Scale bar: 10μm. D) Representative images of mitotic cells stained with anti-tubulin with mitotic defects observed five days after depletion of inner

and outer kinetochore components. Scale bar: 10μm. E) and F) Zoomed-out versions of representative images of cells stained

with anti-tubulin used to classify mitotic defects observed three (E) and five (F) days after depletion of inner and outer

kinetochore components. Scale bar: 30μm.

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Figure S4. Validating the specificity of antibodies raised against CENP-T, Dsn1 and Spc24/25 complex. A) and B) Top:

Schematics showing protein fragments used for antibody generation against lepidopteran CENP-T and Dsn1 homologs.

Bottom: Mass spectrometry results identifying proteins (> 5 peptides) present in immunoprecipitates from B. mori soluble

extracts using the antibody raised against the CENP-T N-terminus or Dsn1, respectively, but not in immunoprecipitates using

the Sigma M2 antibody as a control. C) Top: Schematics showing protein fragments used for antibody generation against the

A)

B)

C)

D) E)

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B. mori Spc24/25 complex. Bottom: Coomassie staining and western blot analyses of different IP fractions pulling down B.

mori His-Spc24/Spc25 protein fragments expressed in Sf9 cells over Nickel-NTA columns. Two bands of the expected size for

the His-Spc24 and Spc25 fragments are labelled with a star on the coomassie-stained gel. Increased concentrations of

immidazol are indicated in mM for the wash and each elution step. The α-His tag immunosignal confirms the presence of the

His-Spc24 fragment in several elution fractions. A band of the same size in the same elution fractions is also recognized by the

custom-made antibody against B. mori Spc24/Spc25 fragments. An additional, much fainter, band corre- sponding to the size

of the B. mori Spc25 fragment is also detected. D) Immunofluorescence signals for CENP-T, Dsn1 and Spc24/Spc25 in

combination with histone 3 serine 10 phosphorylation (H3S10p) in mitotic BmN4 cells. Images from this panel are the same

as in Figure 3. White bar represents the scale of 10 μm. E) Immunofluorescence signals of CENP-T in BmN4 cells transfected

with B. mori CENP-T-GFP-LacI, ΔN-CENP-T-GFP-LacI or LacI-GFP fusion constructs. The anti-CENP-T immunosignal detecting

endogenous CENP-T in interphasic BmN4 cells is low or undetected probably due to the holocentric architecture. In cells

transfected with full-length B. mori CENP-T-GFP-LacI, however anti-CENP-T immunosignal are elevated due to the antibody

recognizing ectopically expressed CENP-T fusion protein. In contrast, in cells transfected with the B. mori ΔN-CENP-T-GFP-LacI,

anti-CENP-T immunosignals are similar to those in control cells expressing LacI-GFP indicating that the N-terminally truncated

CENP-T fusion protein is not recognized by our CENP-T antibody.

117

Figure S5. Depletion of additional outer kinetochore components abolishes recruitment of their respective complex members

in B. mori mitotic cells. A) Representative images of mitotic BmN4-SID1 cells showing the levels of endogenous CENP-T, Dsn1

and Spc24/25 with and without depletion of outer kinetochore components (Mis12, Nsl1 and Spc25). Scale bar: 10 μm. B) Quantification of mean fluorescence intensity for anti-CENP-T, anti-Dsn1 and anti-Spc24/25 signals upon kinetochore

depletion compared to the control. Statistical significance was tested using the Mann and Whitney test (p ≤ 0.0001 for four stars, p ≤ 0.001 for three stars, p ≤ 0.01 for two stars and p ≤ 0.05 for one star).

A)

B)

118

Table S1. Hatchability of the CENP-T mutant strain.

Table S2. Genotypes of hatched larvae from the cross between two heterozygous CENP-T mutants

119

Table S3. Plasmids generated in this study.

120

Table S4. Oligonucleotides used in this study

121

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Annex Cortes-Silva et al., 2020

Article

CenH3-Independent Kinetochore Assembly inLepidoptera Requires CCAN, Including CENP-T

Highlights

d Lepidopteran kinetochores lack CenH3 but contain CCAN

homologs essential for mitosis

d CENP-I depletion impairs Mis12 and Ndc80 complex

recruitment

d CENP-T is sufficient to recruit the Ndc80 and Mis12

complexes

d CENP-T and other CCANs are present in independently

derived CenH3-deficient insects

Authors

Nuria Cortes-Silva, Jonathan Ulmer,

Takashi Kiuchi, ..., Damarys Loew,

Susumu Katsuma, Ines A. Drinnenberg

Correspondence

[email protected]

In Brief

The lepidopteran kinetochore lacks

homologs of CenH3 and CENP-C. Cortes-

Silva et al. describe that CENP-T, a newly

identified kinetochore protein, and other

CCAN homologs are essential for mitotic

progression and kinetochore assembly

and are conserved in independently

derived CenH3-deficient insects.

Cortes-Silva et al., 2020, Current Biology 30, 561–572

February 24, 2020 ª 2019 Elsevier Ltd.

https://doi.org/10.1016/j.cub.2019.12.014

Current Biology

Article

CenH3-Independent Kinetochore Assemblyin Lepidoptera Requires CCAN, Including CENP-T

Nuria Cortes-Silva,1,2 Jonathan Ulmer,1,2 Takashi Kiuchi,3 Emily Hsieh,4,5,6 Gaetan Cornilleau,1,2 Ilham Ladid,1,2

Florent Dingli,7 Damarys Loew,7 Susumu Katsuma,3 and Ines A. Drinnenberg1,2,8,*1Institut Curie, PSL Research University, CNRS, UMR3664, 75005 Paris, France2Sorbonne Universit�e, Institut Curie, CNRS, UMR3664, 75005 Paris, France3Department of Agricultural and Environmental Biology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi

1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan4Molecular and Cellular Biology Graduate Program, University of Washington, Seattle, WA 98195, USA5Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA6Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA7Laboratoire de Spectrom�etrie de Masse Prot�eomique, Institute Curie, PSL Research University, 75005 Paris, France8Lead Contact

*Correspondence: [email protected]

https://doi.org/10.1016/j.cub.2019.12.014

SUMMARY

Accurate chromosome segregation requires assem-

bly of the multiprotein kinetochore complex at

centromeres. In most eukaryotes, kinetochore as-

sembly is primed by the histone H3 variant CenH3

(also called CENP-A), which physically interacts

with components of the inner kinetochore constitu-

tive centromere-associated network (CCAN). Unex-

pectedly, regarding its critical function, previous

work identified that select eukaryotic lineages,

including several insects, have lost CenH3 while

having retained homologs of the CCAN. These find-

ings imply alternative CCAN assembly pathways in

these organisms that function in CenH3-indepen-

dent manners. Here we study the composition and

assembly of CenH3-deficient kinetochores of Lepi-

doptera (butterflies and moths). We show that

lepidopteran kinetochores consist of previously

identified CCAN homologs as well as additional

components, including a divergent CENP-T homo-

log, that are required for accurate mitotic progres-

sion. Our study focuses on CENP-T, which we found

to be sufficient to recruit the Mis12 and Ndc80 outer

kinetochore complexes. In addition, CRISPR-medi-

ated gene editing in Bombyx mori establishes an

essential function of CENP-T in vivo. Finally, the

retention of CENP-T and additional CCAN homologs

in other independently derived CenH3-deficient in-

sects indicates a conserved mechanism of kineto-

chore assembly between these lineages. Our study

provides the first functional insights into CCAN-

based kinetochore assembly pathways that function

independently of CenH3, contributing to the

emerging picture of an unexpected plasticity to

build a kinetochore.

INTRODUCTION

The centromere is an essential chromosomal region that ensures

equal partitioning of chromosomal DNA during cell division [1]. In

all eukaryotes, faithful chromosome segregation requires each

chromosome to interact accurately with microtubule fibers

from the mitotic or meiotic spindle. This interaction is mediated

by the kinetochore, a macromolecular protein complex that as-

sembles on centromeric DNA [2]. The centromere-proximal inner

kinetochore hosts components of the constitutive centromere-

associated network (CCAN), a group of up to 16 different

proteins present throughout the cell cycle that create the centro-

mere-kinetochore interface. Upon cell division, the CCAN re-

cruits the centromere-distal outer kinetochore complex, which

is composed of the KMN network (Knl1, Mis12, and Ndc80 com-

plex) [3]. This recruitment is enabled by two CCAN subunits,

CENP-C and CENP-T, that physically interact with subunits of

the Mis12 and Ndc80 complexes [4–11]. The KMN network

then mediates the interaction with spindle microtubules to drive

chromosome segregation during cell division [12].

Previous work identified the histone H3 variant CenH3 (first

identified as CENP-A in mammals [13, 14]) as a core constituent

for defining the site of a functional kinetochore in most eukary-

otes. This is because CenH3 forms specialized nucleosomes

preferentially found at centromeres that are the target sites for

kinetochore assembly [15–18]. To do so, CenH3 physically inter-

acts with CCAN components, including CENP-C and CENP-N,

connecting the kinetochore to chromatin [19–22]. In addition,

attachment of the kinetochore to chromatin can also be facili-

tated by other CCAN components with DNA-binding activities.

In vertebrates and fungi, this includes CENP-C, the direct DNA

binding partner of CenH3, and the CENP-T/CENP-W complex,

two histone-fold proteins that can form a tetramer with the

CENP-S/CENP-X histone-fold dimer [23–25].

Unexpected to its conservedandessential function,CenH3has

been lost in a fewselect lineages. These include the kinetoplastids

[26, 27],whichhaveevolvedanentirelydifferent kinetochore com-

plex, and an early-diverging fungus with mosaic centromeres

characteristic of both regional and point centromeres [28]. In

Current Biology 30, 561–572, February 24, 2020 ª 2019 Elsevier Ltd. 561

addition, our previous studies also revealed the recurrent loss

of CenH3 in multiple insect species [29]. Interestingly, these spe-

cies have also undergone changes in their centromeric architec-

ture inwhich each lineage independently transitioned frommono-

centric chromosomes (where microtubules attach to a single

chromosomal region) to holocentric chromosomes (wheremicro-

tubules attach along the entire length of the chromosome). This

discovery suggests that these insect species employ an alterna-

tive kinetochore assembly mechanism that occurs chromo-

some-wide in a CenH3-independent manner. Interestingly,

despite the loss of CenH3, computational predictions revealed

the presence of several CCAN and KMN homologs in holocentric

insects [29]. Although those predictions provided some insights

into the composition of these CenH3-deficient kinetochores,

key aspects of kinetochore function, including how these

kinetochores attach to centromeric chromatin and to the outer

kinetochore proteins, remained unresolved.

To address these unknowns, we performed proteomics ana-

lyses in cell lines from the holocentric Lepidoptera (butterflies

and moths). This revealed the presence of several previously un-

identified kinetochore components in these insects, including a

homolog of CENP-T, a core kinetochore component bridging

centromeric DNA to the outer kinetochore in vertebrates and

fungi. We show that, in Lepidoptera, CENP-T and other kineto-

chore components are essential for chromosome segregation

and recruitment of the KMN network. Furthermore, we show

that homologs of CENP-T are also present in other indepen-

dently derived CenH3-deficient insects, indicating a conserved

mechanism of kinetochore assembly between these CenH3-

deficient lineages.

RESULTS

Identification of Kinetochore Components, Including

CENP-T, in CenH3-Deficient Lepidoptera

To gain insights into the composition of CenH3-independent

kinetochores, we performed immunoprecipitation (IP) experi-

ments of kinetochore components we identified previously

in our computational survey [29] (Figure 1A). For this, we es-

tablished several stable Spodoptera frugiperda (Sf9) cell

lines expressing 33FLAG-tagged CCAN (CENP-M, CENP-N,

and CENP-I) and outer kinetochore (Dsn1 and Nnf1) compo-

nents to map their protein interaction profiles by mass

spectrometry.

Mass spectrometry analyses of the kinetochore immunopre-

cipitates recovered all of the previously predicted homologs

of outer kinetochore and inner kinetochore components, con-

firming protein complex formation even in organisms that have

lost CenH3 (Figure 1B; Figure S1A; Data S1). More importantly,

our IPs also revealed several additional components, some of

which harbor remote homology to other known kinetochore

components (Figure 1B). Among those, we identified a CENP-

K-like protein, experimentally supporting previous homology

predictions in Bombyx mori [31]. We also identified two compo-

nents (GSSPFG00019785001 and GSSPFG00001205001) with

remote similarities to the coiled-coil and RWD (RING finger-con-

taining proteins, WD-repeat-containing proteins, and yeast

DEAD [DEXD]-like helicases) domains of the budding yeast

CENP-O and CENP-P proteins, respectively. Furthermore, we

identified the homolog of the a subunit of the ATP synthase (bell-

wether, GSSPFG00010096001), found previously to localize to

kinetochores during Drosophila melanogaster male meiosis

[32], indicating that this function might be extended to lepidop-

teran mitosis.

Both inner and outer kinetochore immunoprecipitates also re-

vealed the presence of a 191.2-kDa protein in S. frugiperda

(GSSPFG00025035001), with KWMTBOMO06797 being the

B. mori homolog (Figure 1B). Iterative hidden Markov model

(HMM) profile searches within annotated proteomes first re-

vealed homologs in several other insect orders and then known

vertebrate CENP-T homologs as best hits (Figure S2A). Similarly,

HMM profile searches within known protein structures identified

the knownGallus gallusCENP-T as the best hit (Figure S2B). The

alignment between the lepidopteran and vertebrate CENP-T

proteins was restricted to their C-terminal parts that include

the histone fold domain (HFD). Importantly, the alignment

extended to the known 2-helix CENP-T extension shown to

interact with the CENP-H/I/K complex in yeast [33], with

several conserved amino acids being identical, supporting

homology to CENP-T rather than to any other histone fold

protein (Figure 1C; Figure S2C). The sequence aligning with the

CENP-T extension appears to be even better conserved

than the HFD, allowing identification of G. gallus CENP-T in

reciprocal HMM profile searches (Figure S2B). Furthermore,

analyses of the primary amino acid sequence revealed

additional similarities between the G. gallus CENP-T and the

B. mori protein, including enrichment of positively charged

amino acids at the N terminus and a proline patch N-terminal

of the HFD (Figure 1C). Finally, reciprocal IP experiment of

the S. frugiperda homolog confirmed its interaction with kineto-

chore components (Figure 1B). Although orthology between

the lepidopteran proteins and CENP-T cannot be confirmed

(Figure S2D), the common sequence architecture, kinetochore-

protein interaction, and role in outer kinetochore recruitment

(see below) lead us to refer to this lepidopteran component as

CENP-T.

Interestingly, we could not detect a putative homolog of the

CENP-T histone fold binding partner CENP-W in any of our IP

experiments, including immunoprecipitates of a 33FLAG-

tagged C-terminal truncated version of CENP-T that contains

the HFD and 2-helix extension (Figure S1B). Our ability to iden-

tify all other known kinetochore homologs in our kinetochore

IPs supports the idea that the inability to identify a lepidopteran

CENP-W homolog is not due to our IP protocol. However, lim-

itations in detecting those potentially small, arginine-rich pro-

teins by mass spectrometry or incomplete annotations cannot

be excluded.

Taken together, we identified previously unknown kineto-

chore components in the CenH3-deficient kinetochore of

Lepidoptera. This includes putative homologs of known

CCAN components, including CENP-T. These results

reinforce and extend our previous computational predictions

indicating similar kinetochore components in Lepidoptera

as those in vertebrates and fungi, despite the loss of

CenH3 in the former. Among those, the identification of

CENP-T was of particular interest because of its known

function in DNA binding and outer kinetochore recruitment in

other organisms.

562 Current Biology 30, 561–572, February 24, 2020

CENP-T and Other Kinetochore Components Are

Required for Accurate Mitotic Progression

In other organisms, depletion of kinetochore components re-

sults in mitotic defects [34]. Previous studies in B. mori

have shown that, consistently, depletion of outer kinetochore

components also results in mitotic defects and increased

numbers of aneuploid cells [35]. We extended this previous

study by using RNAi to deplete a broader catalog of

kinetochore components. Namely, we depleted several outer

kinetochore components of the Mis12 complex (Dsn1,

Mis12, and Nsl1) and the Ndc80 complex (Spc24 and

Spc25) as well as several inner kinetochore components

(CENP-T, CENP-I, CENP-M, and CENP-N). We characterized

the resulting mitotic phenotypes by immunofluorescence (IF)

at two time points (3 and 5 days). We further validated the

efficiency of our kinetochore mRNA depletions by RNA blot

analyses (Figure S3A).

RNAi-mediated depletion of all kinetochore proteins tested re-

sulted in an increase in the number of mitotic cells relative to the

control (RNAi targeting GFP), indicating that kinetochore-

depleted cells are delayed or arrested in mitosis. We observed

the strongest increase of mitotic cells upon CENP-T, CENP-I,

and outer kinetochore depletion, whereas milder enrichment

was seen upon CENP-M and CENP-N depletion (Figure 2B).

Interestingly, although still above control levels, the mitotic

indices upon CENP-I, Spc24, and Spc25 depletion were strongly

decreased at a later time point (Figure S3B), suggesting that,

possibly, cells can exit from mitosis and continue progressing

through the cell cycle or undergo apoptosis.

In addition to the elevated mitotic indices, depletion of

kinetochore components results in various degrees of

mitotic defects in these cells (Figures 2C and 2D; Figures

S3C–S3F). Here, approximately one-fourth of CENP-T-

depleted mitotic cells displayed misaligned monopolar

A B

C

Figure 1. Identification of Kinetochore Components, Including CENP-T, in CenH3-Deficient Lepidoptera

(A) Schematic kinetochore organization in vertebrates and fungi. Boxes indicate subcomplexes within inner and outer kinetochores. Kinetochore components

present in Lepidoptera are highlighted in bold ([29], this study). Kinetochore components that cannot be identified or with limited homology are shown in gray.

(B) The table lists the number of peptides and coverages (in parentheses) of known kinetochore homologs or S. frugiperda proteins identified by mass spec-

trometry that were enriched in at least three of the kinetochore IPs over the control. Samples were boiled from the beads, and the analyses were performed on the

entire sample. The corresponding homologs in B. mori and descriptions based on homology predictions are listed alongside. See also Data S1 and Figure S1.

(C) Top: graphical representation of the B. mori CENP-T and G. gallus CENP-T sequence features, showing the location of the HFDs (blue box), CENP-T family-

specific extension (green box), and arginine-rich (E value 4.73 10�5 and E value 6.03 10�7, respectively) and proline-rich regions (E value 3.73 10�4 and E value

3.4 3 10�8, respectively) (orange and purple boxes, respectively). Bottom: multiple alignment of the C-terminal extension of vertebrate and insect CENP-T

proteins. B. mori (top) andG. gallus (bottom) secondary structure predictions are derived from Jpred4 (a-helical regions are shown in red and b strands in yellow)

[30]. Background coloring of the residues is based on the ClustalX coloring scheme. Full-length insect CENP-T sequences and accession numbers, where

available, are listed in Table S1. See also Figure S2.

Current Biology 30, 561–572, February 24, 2020 563

chromosomes (where one or several chromosomes are not

aligned at the metaphase plate and remain at the spindle

pole). In addition, 34% of CENP-T-depleted mitotic cells

showed complete failure of chromosomes to congress at the

metaphase plate (Figures 2C and 2D). This phenotype was

evenmore pronounced uponCENP-I, Spc24, and Spc25 deple-

tion, with the majority of cells (73%, 76%, and 66%, respec-

tively) unable to successfully congress chromosomes (Figures

2C and 2D). In contrast, mitotic defects upon CENP-M and

CENP-N depletion (Figures 2C and 2D) were relatively mild

but became more pronounced at the later time point (Figures

S3D–S3F). These results show that all tested inner and outer

kinetochore components are required for high-fidelity chromo-

some segregation in B. mori cells. Additionally, the defects

observed upon depletion of the newly identified CENP-T further

support its role in chromosome segregation, which we aimed to

analyze further.

The CENP-T N- and HFD-Containing C Termini Are

Essential for Accurate Mitosis

We next tested whether we could rescue CENP-T knock-

down phenotypes by complementation with an RNAi-

resistant version of CENP-T. Using double-stranded RNA

(dsRNA) targeting the endogenous CENP-T transcript and a

FLAG-tagged recoded CENP-T construct unable to be tar-

geted by the dsRNA, we were able to selectively deplete

B. mori cells of endogenous CENP-T but not FLAG-

tagged CENP-T (Figure S4). These experiments were more

qualitative than quantitative because of the low transfection

efficiency in lepidopteran cells in combination with low

A

B

D

C

Figure 2. Depletion of Kinetochore Components Affects Mitotic Progression in B. mori Cells

(A) Schematic of the RNAi-mediated depletion strategy. BmN4-SID1 [36] cell lines engineered for properties of enhanced uptake of dsRNA were incubated with

various dsRNA constructs for targeted depletion of kinetochore transcripts. After 3 and 5 days, cells were analyzed by IF staining against anti-tubulin and anti-

phospho histone H3-Ser10 (H3S10ph) in combination with each of our custom-made antibodies against the kinetochore protein of interest. The growth medium

was changed on day 3 to add new dsRNA. The efficiency of kinetochore transcript depletion was confirmed by RNA blot analyses (Figure S3A).

(B) Graph showing the percentage of mitotic cells (H3S10ph-positive cells) 3 days after RNAi-mediated depletion of targeted kinetochore components

(n = number of cells ± SEM). For results on day 5, see Figure S3B.

(C) Representative images of mitotic cells stained with anti-tubulin (green), used to classify mitotic defects observed 3 days after depletion of select kinetochore

components. Scale bar, 10 mm. For results on day 5 and depletion of additional outer kinetochore components, see Figures S3C and S3D. For a zoomed-out

version, see Figures S3E and S3F.

(D) Percentages of cells showing no defects (gray), monopolar chromosomes (blue), congression defects (red), and both defects (combination, purple)

(n = number of cells analyzed per condition) for different kinetochore depletion experiments.

564 Current Biology 30, 561–572, February 24, 2020

A

B

C

Figure 3. Depletion of CCAN Components Affects CENP-T and Outer Kinetochore Recruitment in B. mori Cells

(A) Representative images of mitotic BmN4-SID1 cells, showing the levels of endogenous CENP-T, Dsn1, and Spc24/Spc25 with and without depletion of inner

(CENP-T, CENP-I, CENP-M, and CENP-N) and outer (Dsn1 and Spc24) kinetochore components. Scale bar, 10 mm.

(legend continued on next page)

Current Biology 30, 561–572, February 24, 2020 565

numbers of mitotic cells that could be analyzed. Still,

among the cells that could be analyzed, expressing the full-

length RNAi-resistant construct (CENP-Tres-FLAG) rescued

the mitotic defects described above (Figure S4). In contrast,

cells expressing the wild-type, non-resistant construct

(CENP-T-FLAG) displayed mitotic defects, as observed

previously (Figure 2). To evaluate whether the N- or HFD-con-

taining C terminus of the CENP-T protein or both are required

for its function, we expressed RNAi-resistant FLAG-tagged

N- and C-terminally truncated CENP-T constructs (DN-

CENP-Tres-FLAG and DC-CENP-Tres-FLAG, respectively).

Cells expressing either of the two constructs failed to rescue

the observed mitotic defects (Figure S4), leading to the

conclusion that the full-length CENP-T protein is necessary

for accurate mitosis.

Accurate Localization of CENP-T Is Dependent on Other

Inner Kinetochore Components and Is Necessary to

Recruit the Mis12 Complex

Next, to evaluate the role of CCAN and outer kinetochore com-

ponents in kinetochore assembly, we depleted individual kineto-

chore proteins and stained with custom-made antibodies

against the lepidopteran CENP-T, Dsn1 (Mis12 complex), and

Spc24/Spc25 (Ndc80 complex). We validated the specificity of

the antibodies by mass spectrometry, western blot analyses,

and IF upon RNAi-mediated depletion of the respective genes

(Figure 3A; Figure S5).

As expected for kinetochore components, we observed the

immunosignals of CENP-T, Dsn1, and Spc24/Spc25 co-local-

izing with mitotic chromosomes (H3S10ph-positive cells) in

control BmN4 cells (RNAi targeting GFP) (Figure 3A). We then

(B) Quantification of mean fluorescence intensity for CENP-T, Dsn1, and Spc24/25 signals in the control or upon kinetochore depletion. Statistical significance

was tested using a Mann-Whitney test (****p % 0.0001, ***p % 0.001, **p% 0.01, *p % 0.05). For depletion of additional outer kinetochore components, see

Figure S6.

(C) Table summarizing the centromeric localization results of endogenous CENP-T, Dsn1, and Spc24/25 upon RNAi depletion. ++ represents control levels, +

represents reduced fluorescence levels compared with control levels, and – refers to undetected levels.

See also Figures S4 and S5.

A

B

Figure 4. Ectopic CENP-T Is Sufficient to Recruit the Outer Kinetochore in B. mori Cells

(A) Representative images showing localization patterns of endogenous CENP-T, Dsn1, and Spc24/Spc25 (gray) in BmN4-LacO cells transiently expressing LacI-

GFP (top row), CENP-T-GFP-LacI (center row), and DN-CENP-T-GFP-LacI (bottom row) constructs (green). Scale bars, 10 mm.

(B) Quantifications of mean fluorescence intensity of CENP-T, Dsn1, and Spc24/Spc25 at GFP foci versus CENP-T, Dsn1, and Spc24/Spc25 at endogenous sites

(n = 10 cells analyzed). The ratios of mean fluorescence intensities on GFP foci over endogenous loci are shown. Statistical significance was tested using aMann-

Whitney test (****p% 0.0001, **p% 0.01, *p% 0.05).

See also Figure S5.

566 Current Biology 30, 561–572, February 24, 2020

quantified the intensity of the immunosignals on mitotic chromo-

somes in control and kinetochore-depleted cells (Figure 3B).

Depletion of CENP-I, CENP-M, and CENP-N significantly

impaired CENP-T localization to mitotic chromatin. In contrast,

in cells depleted for outer kinetochore components (Dsn1,

Mis12, Nsl1, Spc24, and Spc25), the CENP-T immunosignal

could still be observed (Figures 3B and 3C). In addition, depletion

of any CCAN component resulted in loss of Dsn1. In contrast,

recruitment of Spc24/Spc25, although reduced upon other

CCAN depletion, was only completely abolished upon depletion

of CENP-I (Figures 3B and 3C), consistent with its severe chro-

mosome alignment defects (Figure 2). Dsn1 and Spc24/Spc25

localization was also abolished in cells depleted of other

members of their respective complexes (Figures 3B and 3C; Fig-

ure S6). These data suggest that, in B. mori, recruitment of

CENP-T is dependent on other inner kinetochore components.

In turn, CENP-T appears to be required to recruit the Mis12

complex.

Targeting CENP-T to Ectopic Sites Recruits the Ndc80

and Mis12 Outer Kinetochore Complexes

Our next aim was to evaluate whether CENP-T is sufficient to

recruit outer kinetochore components. For this, we used

B. mori cells transfected with a plasmid containing Lac operator

(LacO) arrays, which enables targeting of transiently expressed

CENP-T-GFP-LacI protein or control (LacI-GFP) constructs

to these loci. To test for the presence of kinetochore compo-

nents, we stained the cells with our custom-made antibodies

against the N terminus of CENP-T, Dsn1, and Spc24/Spc25.

We selected equal-sized areas over the GFP foci and over

mitotic chromosomes and calculated the ratio of their immuno-

signals to determine recruitment of kinetochore proteins to

the LacO array.

As expected, we observed elevated CENP-T immunosignals

over the GFP foci compared to endogenous loci in cells express-

ing full-length B. mori CENP-T-GFP-LacI. In addition, we also

observed elevated Dsn1 and Spc24/Spc25 immunosignals

over the GFP foci, indicating that CENP-T is capable of recruiting

both the Mis12 and Ndc80 outer kinetochore complexes. In

contrast, in cells expressing LacI-GFP, neither CENP-T, Dsn1,

nor Spc24/Spc25 immunosignals are enriched over the LacI-

GFP foci (Figure 4).

In cells expressing the DN-CENP-T-GFP-LacI fusion protein

that is unable to be recognized by our CENP-T antibody (Fig-

ure S5E), we did not measure elevated CENP-T immunosignals

over the GFP foci, indicating that endogenous CENP-T is not re-

cruited to the LacO array. The ratios of Dsn1 and Spc24/Spc25

immunosignals were reduced compared to those in cells ex-

pressing the full-length CENP-T fusion proteins, which indicates

that the N terminus of CENP-T contributes to the recruitment of

these outer kinetochore complexes (Figure 4). Given this, we

conclude that CENP-T is sufficient for the recruitment of the

Ndc80 and Mis12 outer kinetochore complexes and that its N

terminus appears to be important for this activity.

CENP-T Is Essential In Vivo

We next aimed to evaluate the importance of CENP-T in vivo.

For this, CRISPR/Cas9-mediated gene editing was applied to

introduce mutations into the endogenous CENP-T gene of

the B. mori N4 reference strain. A mutant (+7) was isolated

that contains a 7-bp insertion within the guide RNA target

site, causing a premature stop codon in the CENP-T gene (Fig-

ure 5A). Because the C terminus of B. mori CENP-T containing

the HFD and CENP-T extension appears to be essential for its

function (Figure S4), the premature stop codon leads to a non-

functional protein product. Heterozygous CENP-T mutants can

be readily propagated, but when crossed to each other, the

proportion of unhatched eggs increased to about 30%

compared with control crosses (Figure 5B). Genotypic ana-

lyses of the progeny of this cross coming from 70% of eggs

that hatched did not reveal any homozygous CENP-T mutants

(Figure 5C). These results show that, as in vitro, CENP-T is also

essential for B. mori in vivo.

Homologs of CENP-T Are Retained in Independently

Derived CenH3-Deficient Insects

Having characterized the function and importance of CENP-T

for CenH3-independent kinetochore formation in Lepidoptera,

we next profiled its conservation across other CenH3-deficient

and CenH3-encoding insects. Orthologs of the lepidopteran

CENP-T protein can be readily identified using BLASTP in

all CenH3-deficient insects analyzed as well as several

A

B C

Figure 5. The B. mori CENP-T Protein Is Essential In Vivo

(A) CRISPR/Cas9-introduced mutation in the Cenp-T coding sequence. Top:

alignment betweenwild-type (WT) andmutant (+7) sequences, showing a 7-bp

insertion of the Cenp-T gene. Bottom: schematic of WT and truncated protein

products in the CRISPR mutant. The PAM site (red), premature stop codon

(blue), and Cas9 cleavage site (red arrow) are indicated.

(B) Hatching rate of the WT and Cenp-T mutant strains. The percentages of

hatched (gray), unhatched (green), and eggs that died prior to hatching (black)

are indicated for the different crossing patterns between WT (+/+) and het-

erozygous Cenp-T mutants (+/knockout [KO] or KO/+). The number of inde-

pendent crosses (n) is shown above. The raw data are shown in Table S2.

(C) Percentages of genotypes from larvae derived from the cross between two

heterozygous Cenp-T mutants. The number (n) of analyzed larvae is indicated.

The raw data are shown in Table S3.

Current Biology 30, 561–572, February 24, 2020 567

CenH3-encoding insects (Figure 6; Table S1). Notably, phylo-

genetic analyses of HFD proteins belonging to various HFD

families, including CENP-T, indicated accelerated rates of

CENP-T protein sequence evolution ancestral to all insects

(Figure S2D). Importantly, the retention of CENP-T homologs

in independently derived CenH3-deficient insects orders indi-

cates important roles of CENP-T in kinetochores in these

organisms.

DISCUSSION

This study provides new insights into the plasticity of kineto-

chore formation. CenH3 has long been thought to be the

cornerstone of kinetochore formation by mediating the attach-

ment of the kinetochore to chromatin. Its direct DNA binding

partner CENP-C, in turn, contributes to the assembly of other

CCAN components and recruitment of the outer kinetochore

in mitosis [5–7, 20, 21, 37]. In addition to these two central

components, CENP-T emerged as another core component

of the kinetochore, capable of bridging chromatin to the outer

kinetochore complex in vertebrates and fungi [8, 9, 24]. Here

the discovery and characterization of CENP-T in addition to

analyses of other CCAN components in CenH3-deficient Lepi-

doptera provides first insights into alternative pathways to

build a CCAN-based inner kinetochore in a CenH3-indepen-

dent manner.

Our assays using CENP-T artificial tethering indicate that

the role of the lepidopteran CENP-T in outer kinetochore

recruitment might be similar to that in vertebrates and

fungi. In vertebrates, CENP-T is sufficient for recruitment of

both the Mis12 and the Ndc80 complexes by directly interact-

ing with their respective subunits [5, 11, 38]. In fungi, the

CENP-T N terminus directly interacts with the Spc24/Spc25

subunit to recruit the Ndc80 complex [8, 39]. Our results

show that the lepidopteran CENP-T is also sufficient to

recruit the Mis12 and Ndc80 complexes. Whether CENP-T,

in particular its N terminus, makes direct protein interactions

with any of the Ndc80 or Mis12 complex subunits remains

to be shown. However, CENP-T might not be the only factor

recruiting outer kinetochore complexes. In fact, though

strongly reduced, Spc24/Spc25 and Dsn1 are still present

on DN-CENP-T-GFP-LacI foci, which indicates that

their recruitment is either aided by more internal regions of

CENP-T or via another kinetochore components recruited

by CENP-T. Furthermore, our observation that the recruitment

of the Spc24/Spc25 is only reduced but not completely

abolished upon CENP-T depletion suggests that at least one

additional Ndc80 receptor exists at the kinetochore, perhaps

via CENP-I (see below).

In addition, other CCAN components also appear to have

essential roles in CenH3-independent kinetochore assembly.

For example, we find that, upon depletion of CENP-I, Mis12

A B

Figure 6. Homologs of CENP-T Are Present in All Other CenH3-Deficient Insects

(A) Holocentric insect orders and species are indicated in blue, and inferred multiple transitions to holocentric chromosomes are labeled with ‘‘H.’’ Using protein

homology searches of genomes or assembled transcriptomes, the ability and inability to find CenH3 and CENP-T homologs are indicated by a black or white box,

respectively. Orthologs of insect CENP-T sequences are listed in Table S1. A phylogeny of a set of HFD sequences from various families, including CENP-T, is

shown in Figure S2D, based on HFD sequences listed in Data S2. Although our previous studies did not identify any holocentric insect species that encode for

CenH3 [29], searches in additional organisms revealed the presence of putative CenH3 proteins in water striders (Hemiptera). This result provides new insights

into the transition to CenH3-deficient holocentric architectures in that the change in centromeric architecture appears to precede the loss of CenH3, perhaps by

providing the necessary conditions to allow loss of this otherwise essential component.

(B) Schematic of lepidopteran kinetochore subunits analyzed in this study. Black arrows indicate full localization dependencies. Grey dashed arrows indicate that

localization dependency is unknown.

568 Current Biology 30, 561–572, February 24, 2020

and Ndc80 complex recruitment is completely abolished,

consistent with CENP-I depletion resulting in the strongest

mitotic defects. A role of the lepidopteran CENP-I in recruiting

the Ndc80 complex would recapitulate previous observations

in other organisms showing that the vertebrate CENP-H/I/K/

(M) complex contributes to Ndc80 localization [40], that

the C terminus of human CENP-I localizes closely to Ndc80

[41], and that CENP-H/I/K/(M) subunits directly interact

with Ndc80 in vertebrates and budding yeast [33, 42].

Given these findings together with our CENP-I depletion ana-

lyses, it will be interesting to test whether CENP-I or CENP-I

complex members makes direct protein interactions with

the Ndc80 complex in Lepidoptera and contributes to its

recruitment. Overall, the tools that were generated in this

study will facilitate future studies to dissect the contribution

of CENP-I, other CCANs, or additional kinetochore compo-

nents with unknown evolutionary relationships (Figure 1) to

CenH3-independent kinetochore assembly and chromatin

attachment in Lepidoptera.

Considering the essential roles of CenH3 and CENP-C in all

other organisms tested, the recurrent loss of these proteins in

several insects indicates that the potential for CenH3/CENP-C-

independent kinetochore formation might have already arisen

in an early insect ancestor. Such potential could be repre-

sented in the form of other kinetochore components that

evolved the capability to compensate (partially or completely)

for CenH3/CENP-C-dependent roles in kinetochore-chromatin

attachment and outer kinetochore recruitment. The retention

of CENP-T homologs in independently derived CenH3/CENP-

C-deficient insects suggests their important contributions to

kinetochore assembly, perhaps by contributing CenH3/

CENP-C-mediated functions. Among these, future studies

will, in particular, aim to evaluate the contribution of the lepi-

dopteran CENP-T in kinetochore-chromatin attachment.

Nevertheless, it is also possible that CenH3/CENP-C-medi-

ated functions have been compensated by other CCAN com-

ponents. In fact, several other CCAN components, including

CENP-I and CENP-N, are also retained in CenH3-deficient in-

sects [29], indicating critical roles in their kinetochore assem-

blies as well. Notably, given that D. melanogaster has lost

most CCAN components and solely relies on CenH3 and

CENP-C for inner kinetochore assembly, it is unlikely that

either protein is dispensable for chromosome segregation in

this species.

Our results also exemplify the necessity of experimental

data to obtain comprehensive pictures of kinetochore

complex composition. The characterization of kinetochore

components in additional eukaryotes will enable us to obtain

better insights into the sequence divergence of kinetochore

homologs. This will allow us to increase the information con-

tent of our alignments, improving our homology prediction

capabilities.

Finally, our studies of kinetochore composition of CenH3-

deficient holocentric lepidopteran species, together with the

recent finding of a CenH3-deficient monocentric fungus [28,

43] that encodes for other CCAN components, including

CENP-T, show that CCAN-based CenH3-independent kineto-

chore assemblies are considerably widespread in diverse

eukaryotes.

STAR+METHODS

Detailed methods are provided in the online version of this paper

and include the following:

d KEY RESOURCES TABLE

d LEAD CONTACT AND MATERIALS AVAILABILITY

d EXPERIMENTAL MODEL AND SUBJECT DETAILS

B Lepidopteran cell lines and culture conditions

B Culture conditions of B. mori N4 strain

d METHOD DETAILS

B Alignments and phylogenetic analyses

B Homology predictions

B Plasmid construction

B Construction of cell lines

B Protein expression and affinity purification

B Validation of CENP-T and Dsn1 antibody specificity

B Validation of B. mori Spc24/25 antibody specificity

B Affinity co-immunoprecipitations

B Proteomics and Mass Spectrometry Analysis

B Immunofluorescence

B Microscopy

B RNAi-mediated knock-down

B RNA blot analyses

B CRISPR-mediated genome editing in B. mori

d QUANTIFICATION AND STATISTICAL ANALYSIS

d DATA AND CODE AVAILABILITY

SUPPLEMENTAL INFORMATION

Supplemental Information can be found online at https://doi.org/10.1016/j.

cub.2019.12.014.

ACKNOWLEDGMENTS

We thank Harmit S. Malik and Steve Henikoff for support during the initial steps

of this project; Alexander Schleiffer and Eelco Tromer for helpful discussions

regarding protein homology predictions; Alexander Schleiffer for sharing a

collection of representative histone fold proteins;, Takahiro Kusakabe for the

BmN4-SID1 cell line; Tatsuo Fukagawa for the CENP-T-FLAG DT40 cell line

we used for control experiments; Abderrahman Khila for access to water-

strider genome sequences; Patricia Le Baccon, Mickael Garnier, and Solene

Herv�e for help with microscopic analyses; Ahmed El Marjou and Carlos Kikuti

for help and advice regarding protein purification and biochemistry; Carsten

Janke for advice regarding microtubule staining; all members of the Fachinetti

and Drinnenberg labs for helpful discussions; and Leah Rosin and all members

of theDrinnenberg lab for input on themanuscript. N.C.-S. receives salary sup-

port from Sorbonne Universit�e (2781/2017). This work was supported by

‘‘R�egion Ile-de-France’’ and Fondation pour la Recherche M�edicale grants

(to D.L.). This work was supported by grants from JSPS KAKENHI

(15H02482 to S.K. and T.K.). I.A.D. and I.L. receive salary support from the

CNRS. This work was supported by LabEx DEEP (ANR-11-LABX-0044,

ANR-10-IDEX-0001-02), an ATIP-AVENIR research grant, Institut Curie, and

the ERC (CENEVO-758757).

AUTHOR CONTRIBUTIONS

N.C.-S. performed and analyzed the cell biological experiments. J.U. and E.H.

generated cell lines and performed the IP experiments. T.K. generated and

analyzed the B. mori CENP-T knockout strain. S.K. supervised the in vivo an-

alyses. I.A.D. performed the computational and phylogenetic analyses. G.C.

and I.L. generated constructs and helped with the biochemical and cell

biological experiments. F.D. carried out the MS experimental work. D.L.

Current Biology 30, 561–572, February 24, 2020 569

supervised MS and data analysis. I.A.D. supervised the study and secured

funding. N.C.-S. and I.A.D. wrote the manuscript.

DECLARATION OF INTERESTS

The authors declare no competing interests.

Received: November 2, 2019

Revised: December 3, 2019

Accepted: December 5, 2019

Published: February 6, 2020

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80. Ota, S., Hisano, Y., Muraki, M., Hoshijima, K., Dahlem, T.J., Grunwald,

D.J., Okada, Y., and Kawahara, A. (2013). Efficient identification of

TALEN-mediated genome modifications using heteroduplex mobility as-

says. Genes Cells 18, 450–458.

81. Ansai, S., Inohaya, K., Yoshiura, Y., Schartl, M., Uemura, N., Takahashi, R.,

and Kinoshita, M. (2014). Design, evaluation, and screening methods for

efficient targetedmutagenesis with transcription activator-like effector nu-

cleases in medaka. Dev. Growth Differ. 56, 98–107.

82. Vizcaıno, J.A., Csordas, A., Del-Toro, N., Dianes, J.A., Griss, J., Lavidas, I.,

Mayer, G., Perez-Riverol, Y., Reisinger, F., Ternent, T., et al. (2016). 2016

update of the PRIDE database and its related tools. Nucleic Acids Res. 44,

11033.

572 Current Biology 30, 561–572, February 24, 2020

STAR+METHODS

KEY RESOURCES TABLE

REAGENT or RESOURCE SOURCE IDENTIFIER

Antibodies

Mouse monoclonal Anti-FLAG M2 antibody Sigma Cat#F1804; RRID: AB_262044

Rabbit polyclonal anti-Spc24/25 (rabbit 1621016) This study N/A

Rabbit polyclonal anti-CENP-T (rabbit 045) This study N/A

Rabbit polyclonal anti-Dsn1 (rabbit 1615031) This study N/A

Mouse monoclonal anti-6xHis Sigma Cat#ab18184; RRID:AB_444306

Goat monoclonal IRDye 680RD anti-Rabbit IgG LI-COR Cat#926-68071; RRID:AB_10956166

Donkey monoclonal IRDye 800CW anti-mouse IgG LI-COR Cat#926-32212; RRID:AB_621847

Mouse monoclonal anti-FLAG M2 beads Sigma Cat#M8823; RRID:AB_2637089

Mouse monoclonal anti-a-tubulin Alexa Fluor 488 Thermo Fisher Scientific Cat#53-4502-80; RRID:AB_1210526

Mouse monoclonal anti-FLAG M2 Sigma Cat#F1804; RRID:AB_262044

Rat monoclonal anti-phospho Histone H3-Ser10 Sigma Cat#MABE939

Goat polyclonal anti-rabbit IgG Alexa Fluor 568 Thermo Fisher Scientific Cat#A-11011; RRID:AB_143157

Goat polyclonal anti-rat IgG Alexa Fluor 568 Thermo Fisher Scientific Cat#A-11077; RRID:AB_2534121

Goat polyclonal anti-rat IgG Alexa Fluor 488 Thermo Fisher Scientific Cat#A-11006; RRID:AB_2534074

Goat polyclonal anti-mouse IgG Alexa Fluor 488 Thermo Fisher Scientific Cat#A-11029; RRID:AB_2534088

Goat polyclonal anti-mouse IgG Alexa Fluor 568 Thermo Fisher Scientific Cat#A-11004; RRID:AB_2534072

Goat polyclonal anti-rat IgG Alexa Fluor 633 Thermo Fisher Scientific Cat#A-21094; RRID:AB_2535749

Bacterial and Virus Strains

E. coli Bl21DE30 plys pRare Sigma 71400

B. mori Spc24/Spc25 baculovirus This study N/A

DH10BacLL Ahmed EL-Marjou N/A

Chemicals, Peptides, and Recombinant Proteins

Cellfectin II GIBCO Cat#10362100

cOmplete Protease Inhibitor Cocktail Roche Cat#11697498001

SUMO-Protease Ahmed EL-Marjou N/A

Bolt 4-12% Bis-Tris Plus denaturing gels Invitrogen Cat#NW04120BOX

MNase Sigma Cat#N3755-500UN

FLAG peptide Sigma Cat#F4799

Novex 16% Tris Glycine Precast Gels Invitrogen Cat#XP00162BOX

Magnetic Dynabeads Protein A Invitrogen Cat#10002D

DNase I Roche Cat#04716728001

4-20% Tris glycine gels Invitrogen Cat#XP04200BOX

InstantBlue Sigma Cat#ISB1L

PVDF membrane Bio-Rad Cat#170-4272

Odyssey Blocking buffer LI-COR Cat#927-50000

Trypsin/LysC Mix, Mass Spec Grad Promega Cat#v5071

LabSafe GEL Blue GBiosciences Cat#786-35

NuPAGE 10% Bis-Tris Protein Gels, 1.0 mm, 10-well Invitrogen Cat#NP0301BOX

DTT, dithiothreitol Euromedex Cat#EU0006-B

Iodoacetamide Sigma-Aldrich Cat#I6125

MeCN, acetonitrile Merck Cat#1.00099

HCOOH, formic acid Fluka Cat#94318

(Continued on next page)

Current Biology 30, 561–572.e1–e10, February 24, 2020 e1

Continued

REAGENT or RESOURCE SOURCE IDENTIFIER

NH4HCO3, ammonuim bicarbonate Fluka Cat#09830

DAPI Sigma Cat#D9542

Vectashield Antifade Mounting Medium Vector Laboratories Cat# H-1000; RRID:AB_2336789

Dithiobis(succinimidyl propionate, DSP) Thermo Fisher Scientific Cat#22585

Hank’s balanced salt solution (HBSS) GIBCO Cat#14025050

TRIzol Invitrogen Cat#15596018

NorthernMax-Gly Sample Loading Dye Thermo Fisher Scientific Cat#AM8551

NorthernMax-Gly Gel Running Buffer Thermo Fisher Scientific Cat#AM8678

QuickHyb solution Agilent Cat#201220

Salmon-sperm ssDNA Sigma Cat#31149

Cas9 Nuclease protein NLS NIPPON GENE Cat#319-08641

Critical Commercial Assays

Gateway system Invitrogen Cat#11791019

Branson Digital Sonifier SFX550 Branson Ultrasonics N/A

Protino Ni-TED 1000 columns Machery-Nagel Cat#745110.50

Covaris E220 Evolution ultrasonicator Covaris N/A

Amicon Ultra-0.5 mL 3K MWCO filters Sigma Cat#UFC5003

Pierce Silver Stain Kit Thermo Fisher Scientific Cat#24612

C18 precolumn (300 mm inner diameter x 5 mm) Thermo Fisher Scientific Cat#164942

C18 column (75 mm inner diameter x 50 cm) Thermo Fisher Scientific Cat#164535

Orbitrap Fusion Tribrid mass spectrometer Thermo Fisher Scientific N/A

UltiMate 3000 RSLCnano System Thermo Fisher Scientific N/A

Nanospay Flex ion source Thermo Fisher Scientific N/A

Q Exactive HF-X Thermo Fisher Scientific N/A

TurboBlotter System Bio-Rad Cat#1704155

MAXIscript T7 Transcription kit Thermo Fisher Scientific Cat#AM1312

Tris (HotSHOT) method [44] N/A

KOD One TOYOBO Cat# KMM-101

MultiNA microchip electrophoresis system SHIMADZU N/A

MultiNA microchip electrophoresis system - DNA-500

reagent kit

SHIMADZU Cat# 292-27910-91

BigDye Terminator v3.1 Cycle Sequencing Kit Applied Biosystems Cat#4337454

ABI PRISM 3130xl Genetic Analyzer Applied Biosystems N/A

Deposited Data

Proteomics data This study PRIDE: PXD016092

Experimental Models: Cell Lines

BmN4 ATCC Cat#CRL-8910; RRID: CVCL_Z633

BmN4-SID1 [36] RRID:CVCL_Z091

Sf9 GIBCO Cat#12659017

Sf9-LacO This study N/A

Experimental Models: Organisms/Strains

Non-diapause B. mori strain N4 University of Tokyo N/A

Cenp-T mutant B. mori This study N/A

Oligonucleotides

Oligonucleotides are listed in Table S6 This study N/A

Recombinant DNA

pIBV5 plasmid Invitrogen Cat#12550018

pENTR vector Invitrogen Cat#K240020

(Continued on next page)

e2 Current Biology 30, 561–572.e1–e10, February 24, 2020

LEAD CONTACT AND MATERIALS AVAILABILITY

All the reagents generated in this study are available for sharing. Further information and requests for resources and reagents should

be directed to and will be fulfilled by the Lead Contact, Ines Anna Drinnenberg ([email protected]).

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Lepidopteran cell lines and culture conditions

Cultured silkworm ovary-derived BmN4 (ATCC Cat#CRL-8910; RRID: CVCL_Z633) and BmN4-SID1 cell lines (RRID:CVCL_Z091)

[36] were maintained in Sf-900 II SFM medium (GIBCO Cat#10902-088) supplemented with 10% fetal bovine serum (Eurobio

Cat#CVFSVF0001), antibiotic-antimycotic (GIBCOCat#15240-062) and 2mML-glutamine (GIBCOCat#25030-024) at 27�C. Sf9 cells

(GIBCO Cat#12659017) were maintained in Sf-900 II SFM medium (GIBCO Cat#10902-088) supplemented with antibiotic-antimy-

cotic (GIBCO Cat#15240-062) and 2mM L-glutamine (GIBCO Cat#25030-024) at 27�C.

Culture conditions of B. mori N4 strain

Non-diapaused larvae of the B. mori N4 strain were fed with fresh mulberry leaves or artificial diet SilkMate (NOSAN SilkMate PS)

under a continuous cycle of 12-h light and 12-h darkness at 25�C.

Continued

REAGENT or RESOURCE SOURCE IDENTIFIER

pVS1-LacO [45] Addgene RRID: Addgene_33143

eGFP_N1_LacI pDEST Genevieve Almouzni

lab [46]

N/A

pCMV-lacI Genevieve Almouzni lab

Agilent

Cat#217450

pRS416 [47] N/A

pIZV5 Invitrogen Cat#V800001

pT7-His-SUMO vector Ahmed EL-Marjou N/A

pRSF-DUET1 Sigma Cat#71341

Software and Algorithms

MAFFT [48] https://mafft.cbrc.jp/alignment/software/

Jalview [49] https://www.jalview.org/

Jpred4 [30] http://www.compbio.dundee.ac.uk/jpred/

PhyML 3.0 [50] http://www.atgc-montpellier.fr/phyml/

iTOL vs4 [51] http://itol.embl.de

fLPS [52] http://biology.mcgill.ca/faculty/harrison/flps.html,

or https://github.com/pmharrison/flps

HMMER webserver [53] http://www.ebi.ac.uk/Tools/hmmer

HHpred version 3.2.0 [54] https://toolkit.tuebingen.mpg.de/tools/hhpred

phyre2 predictions [55] http://www.sbg.bio.ic.ac.uk/phyre2/html/

page.cgi?id=index

Proteome Discoverer (v 2.2) ThermoFisher Scientific N/A

myProMS [56] https://github.com/bioinfo-pf-curie/myproms

Fiji [57] http://fiji.sc/

Prism version 8.12 for Mac GraphPad Software https://www.graphpad.com/scientific-software/

prism/

CRISPRdirect [58] https://crispr.dbcls.jp/

Other

S. frugiperda ‘‘corn strain’’ annotation [59] https://bipaa.genouest.org/sp/spodoptera_

frugiperda_pub/

S. frugiperda ‘‘rice strain’’ annotation [59] https://bipaa.genouest.org/sp/spodoptera_

frugiperda_pub/

S. frugiperda transcriptome TR2012b [60] http://bioweb.ensam.inra.fr/spodobase

SilkBase [61] http://silkbase.ab.a.u-tokyo.ac.jp;

RRID:SCR_008242

Current Biology 30, 561–572.e1–e10, February 24, 2020 e3

METHOD DETAILS

Alignments and phylogenetic analyses

For Figure 1C and Figure S2C, sequences were aligned with MAFFT on the EMBL-EBI web interface [62], and visualized and pro-

cessed with Jalview [49] (ClustalX coloring scheme). Secondary structures were predicted using Jpred4 [30]. For Figure S2D, histone

fold domains were aligned using MAFFT [48, 63]. A maximum likelihood phylogeny was build using PhyML 3.0 [50] with automatic

model selection [64], default parameters and 100 bootstrap permutations. The tree was visualized using iTOL vs4 [51]. Sequences

used for the phylogeny are listed in Data S2.

Primary sequence analyses of the B. mori andG. gallus CENP-T (GenBank: NP_001263242.1) for Figure 1C were performed using

fLPS [52] with default parameters. The Arginine-rich N-terminal regions are located between amino acid 4 and 32 of the G. gallus

CENP-T (E value 6.0x10�7); and 20 and 41 of the B. mori CENP-T (E value 4.7x10�5). The proline-rich regions are located between

amino acid 493 and 521 of the G. gallus CENP-T (E value 3.4x10�8); and 850 and 899 of the B. mori CENP-T (E value 3.7x10�4).

Homology predictions

The annotation of the S. frugiperda corn strain proteome were used for all computational analyses [59]. In case annotations were

missing or incorrect we complemented the data using annotations from the S. frugiperda ‘‘rice strain’’ [59], EST (Transcriptome

TR2012b) data [60] or our own analyses. Homologs of S. frugiperda proteins identified in the kinetochore IPs were predicted in

B. mori proteome [61]. Both S. frugiperda and B. mori proteins were used for homology searches. BLASTP and PSI-BLAST searches

were performed in the NCBI non-redundant protein database [65]. Jackhmmer searches were performed on the HMMER webserver

[53] against reference proteomes as current as September 2019. HHpred version 3.2.0 searches were performed against the

PDB_mmCIF70_3_Aug [54]. Coiled-Coil domains were predicted using PCOILS (window 28) [54, 66, 67].

CENP-T

For iterative HMMprofile searches only the C-termini containing the HFD and 2 helix extension of theB.mori or S. frugiperda proteins

were used.

> B_mori_CENPT_CTERM

TTKRLYKYLEDKLEPKYDYKARVRAEKLVETIYHFTKEVKKHEVAPNDAVDVLKHEMARLDIVKTHFDFYQFFHDFMPREIRVKVVPDIVN

KITIPRNGVFSEILSGHAVHA

> S_frugiperda_CENPT_CTERM

ITKRLYKFLETKLEPKYDYKARVRAEKLVETIYHFAKDLRRHDVAPTDAVDVLKHELARLEVVQTHFEFYEFFHEFMPREVRVKVVPDIVN

KIPLPRHGVFSDILRGNNVQG

HHpred searches using the C terminus of the B. mori and S. frugiperda CENP-T protein identify the C terminus of the G. gallus

CENP-T with high probability 94.56% and 94.2% respectively (Figure S2). HHpred searches using the full-length protein also iden-

tifies theG. gallusCENP-T as a best hit thoughwith lower probabilities (51.9% for theB.mori and 50% for theS. frugiperda protein). In

this search, the alignment between the lepidopteran protein andG. gallusCENP-T is restricted to their C-termini. A reciprocal HHpred

search using the G. gallus HFD helix 3 and CENP-T extension identified B. mori protein as best hit (Figure S2).

Known vertebrate CENP-T proteins can also be identified as best hits after 2 jackhmmer iterations using the B. mori CENP-T C

terminus (Figure S2). Additional arthropod CENP-T from the whiteleg shrimp (Penaeus vannamei) and the pill woodlouse (Armadilli-

dium vulgare) could be predicted after 4 jackhmmer iterations. Finally, phyre2 predictions [55] using the full-length B. mori CENP-T

protein predicts the known structure of theG. gallusCENP-T C terminus with high confidence (91.5%) while low sequence identity of

the aligned regions (14%).

Iterative blastp and tblastn searches using lepidopteran CENP-T proteins against select annotated insect proteomes, genome as-

semblies and assembled transcriptomes revealed additional orthologs of CENP-T proteins in insects (Table S1).

KWMTBOMO14835 and GSSPFG00001205001

BLASTP searches using both proteins against the non-redundant protein database revealed only hits in Lepidoptera. Iterative PSI-

BLAST searches and jackhmmer searches using both proteins also revealed no homologous hits in other insect orders. HHpred

searches of GSSPFG00001205001 did not reveal any hits with high probability. However, HHpred searches identified similarity be-

tween the N terminus of KWMTBOMO14835 and resolved structure of the S. cerevisiae Ctf19 protein (CENP-P) [68] as best hit with

high probability (93.58%) while only 13% amino acid identity. The aligned region also includes parts of the tandem RWD domains

from S. cerevisiaeCtf19. The N terminus of KWMTBOMO14835 has predicted coil-coiled regions (PCOILS, window 28). HHpred pre-

dictions of the trimmed protein without the coiled-coil region reduced the probability of similarity to Ctf19 to 43.96%, which was also

no longer the best hit.

We therefore refrain from assigning homology to Ctf19 without further functional or structural validations and refer to the lepidop-

teran proteins as coiled-coil RWD-like proteins.

KWMTBOMO09290 and GSSPFG00019785001

Both proteins contain N-terminal coiled-coil domains (PCOILS, window 28). HHpred searches of both proteins without the N-terminal

coiled-coil regions revealed similarities to RWD domains of several kinetochore components including the resolved structure of the

G. gallus Spc24 globular domain [9] and Mcm21 (CENP-O) subunit of the budding yeast Ctf19 complex [68, 69] with high and

similar probabilities. Iterative jackhmmer searches of the trimmed B. mori protein without the N-terminal coiled-coil region identified

hits in Hymenoptera in the second iteration includingCamponotus floridanus (UniprotKB: E2AEP7, E value 0.0017). The third iteration

e4 Current Biology 30, 561–572.e1–e10, February 24, 2020

identified additional hits in the blattodean Zootermopsis nevadensis (UniprotKB: A0A067RMY5, E value 0.00028), the arthropod

Daphnia magna (UniprotKB: A0A0P5Q3Q5, E value 6.5x10�7) and predicted known vertebrate CENP-O proteins in Anabis testudi-

neus (UniprotKB: A0A3Q1H0S6, E value 0.0023 and UniprotKB: A0A3Q1H0T5, E value 0.0037). The fourth iteration identified the hu-

man CENP-O (E value 1.6x10�13).

Given the similarity to multiple RWD containing kinetochore proteins and the lack of experimental evidence we only refer to the

lepidopteran proteins as coiled-coil RWD-like proteins.

KWMTBOMO06154 and GSSPFG00011797001

HHpred searches did not identify high scoring hits for neither one of the two proteins. GSSPFG00011797001 identified the recently

resolved structure of the fungus Thielavia terrestris CENP-H [70] but not as the best hit and with only 36% probability. Still, given the

presence of CENP-I, CENP-M and the putative CENP-K, it will be worthwhile to test if these proteins are part of a lepidopteran CENP-

H-I-K-M complex. Hits in other insect orders could not be predicted using psi-blast or jackhmmer.

LOC101741561 and GSSPFG00006732001

The B. mori homolog is not part of the most recent annotations [61], we therefore refer to the ID of previous annotations present on

NCBI Genebank in Figure 1.

GSSPFG00006732001 is likely misannotated because most other lepidopteran homologous proteins including GenBank:

LOC101741561 only align to the C-terminal part of the protein. We derived a new consensus sequence by aligning available

TR2012b transcriptome data [60]. The encoded ORF translates into the following protein:

> S. frugiperda CENP-K

MSSDRNKETRDAVKREIKEIQARCKHEWNIIDNSPLDAPNIDLEQINEKALCYIEGLGTGIQNSNTPITADDNLLTSQFLKEIRDKTGQVEE

YTAFVRGSIHDIDAEINRLQTLIKITQEAKARPMLNKCEVQPEHVHRAKERFQVMKNELHSLIHSLFPNCDSLIIETMGQLMAEHLNEESN

GYIPVTAETFQIIELLKDMKIVTVNPYNKLEVKLSY

One of the EST that contain the full ORF is Sf2H05447-5-1 that is listed in Figure 1B.

Iterative PSI-BLAST searches using the B. mori protein revealed hits in Hymenoptera including Athalia rosae (GenBank:

LOC105689000, E value 5x10�5) after the 1st iteration. The 2nd iteration revealed hits in Hemiptera includingBemisia tabaci (GenBank:

LOC109036758, E value 0.003), the Blattodean Zootermopsis nevadensis (GenBank: LOC110834781, E value 2x10�11) and the

mollusk Mizuhopecten yessoensis (centromere protein K-like, E value 0.001). The human CENP-K was identified after the 3rd itera-

tion. These analyses are consistent with previous predictions identifying CENP-K in B. mori [31].

Jackhmmer searches of a N-terminal truncated version of the B. mori protein without a coiled-coil region also revealed a hit in the

phthirapteran Pediculus humanus corporis after the 3rd iteration (UniprotKB: E0VW71, E value 4.1x10�9).

KWMTBOMO11351 and GSSPFG00010096001

These are orthologs ofDrosophila melanogaster bellwether, the alpha subunit F0F1 ATP synthase subunit alpha recently been shown

to interact with Cid during Drosophila melanogastermale meiosis [32]. Orthologs are present across insects and diverse eukaryotes.

KWMTBOMO11557 and GSSPFG00019290001

HHpred searches aligns both proteins to the structure of the CHRAC 14 HFD [71] as best hits but low probabilities (Probability 49.3%

for KWMTBOMO11557 and 39.1% for GSSPFG00019290001). Hits in other insect orders could not be predicted using psi-blast.

Jackhammer searches of both proteins converged after 2 iterations.

Sf2H06980-5-1 and KWMTBOMO014775

The S. frugiperda protein is not part of the S. frugiperda corn strain annotations but in the annotations from the closely-related

S. frugiperda rice strain [59]. We therefore provide the ID from these annotations file. Both B. mori and S. frugiperda proteins contain

C-terminal coil-coiled regions. HHpred searches using only the N-terminal first 65 amino acids identifies the known structure of the

human Nsl1 [72].Though the human Nsl1 was not the best hit for neither one of the two lepidopteran proteins, given the abundance of

the protein in the Nnf1 and Dsn1 IPs we refer to it as Nsl1 candidate. These data suggest that as in other eukaryotes, the lepidopteran

Mis12 complex contains four instead of only three components as recently described [35].

Hemipteran CenH3

Hemipteran CenH3 fragments in Gerris buenoi genome assembly [73] and Aquarius paludum assembly (A. Khila, personal commu-

nication) could be identified in TBLASTN searches using D. melanogaster H3 as queries.

> Aquarius_paludum_Embryo_Assembly50_c84768)g2_il

RRRSGVVALREIRHLQKSTNLLIPKLPFMRIVQEILQSYSTEPYRIQSRALEALQEMTEILMVDLFSEAILCCIHAKRKTIMVQDMRLARRIRG

> Gerris_buenoi_ KZ651887.1

RRRSGVVALREIRHLQKSTNLLIPKLPFMRIVKEILQSYSTEPYRLQTQALEALQEMTEILMVDLFSEAILCCLHAKRKTIMVQDMRLARRIRG

Plasmid construction

A list of plasmids generated in this study is provided in Table S4. For the kinetochore IPs, full-length ORFs of S. frugiperda CENP-M,

CENP-N, CENP-I, CENP-T, Dsn1 andNnf1 fused to 3xFLAG tagswere cloned into pIBV5 (Invitrogen Cat#12550018) using theGateway

system (Invitrogen Cat#11791019) from pENTR vectors (Invitrogen Cat#K240020). The genes encoding for S. frugiperda CENP-T,

CENP-I and CENP-N are not correctly annotated in the current assembly [59] as inferred by aligning the protein products to the

B. mori homologs. We inferred the correct ORF sequences from EST data and sequencing of the amplified PCR fragments from Sf9

cDNA.

Current Biology 30, 561–572.e1–e10, February 24, 2020 e5

The following are the correct ORF sequences that were used for all experimental analyses:

> S. frugiperda CENP-I

MADVDEIIDYIKSLKKGFDKDLFQNKIDELAYAVDTTGILYNDFHTLFKVWLNLSIPITKWVSLGACLVPQNIVEDRTVEYALSWMLSNYED

QSTFSRIGFLLDWLTAAMECECIEMETLDMGYDVFYVSLTYETLTPHAVKLVYTLTKPVDVTRRRVLELLDYARKREAKKNMFRQLXVLL

GLFKSYKPECVPEDIPAISIHAAFKKINPDLLARFKRNQENRNSVRRERHHLTWINPINSDRGRNKKIDPLVPNVEFLNIGSKQYAEKEHQK

NFLDFTDPVSVLQCSVQRSTSRPARIRALLCNVTGVALLAVASHTEQEFLSHDLHHLLNSCFLNISPHSYREKQDLLHRLAVLQHTLMQG

IPVITRFLAQYLPLWNERDYFAEILELVQWVSLDSPDHVTCVLEPLARAYHRAQPIEQCAILRSLNHMYCNLVYASTRKRHHFMGTPPSP

QVYALVLPKVATAISDMCDKELQVNPEQMVVLHSGVQGAAARARGEARGGAAAGALAPRLALALPLLASSAALLDSVAELMILYKKIFTT

AKQTNVITNTDTFEKQMQVLEAYTSDLINCLYSEGALSDRNLGFVFSKLHPQLVEKLGSLMPDVDAKLSIRNSIAFAPYTYIQLDAIDHRD

ADNKLWFNAVIEQEFTNLSRFLKRAVTELRYQ

> S. frugiperda CENP-T

MPKTKIPSPARPQGATTPKRKKSRGSRSVCSPALSTASGRLTSLIDQFKEKNMESFALSPLNRRSILNDDTAIEEPRRQSWWKKLKEDS

HEIMEVLEENKVADSGNNAIEELIDIEVLSQEKKEYTLDLPESSDNESINSIVLPQRKLFTQKENKPQKKFGQFSDNRETLAKLHKTNTQG

DKTVNVGTRELFQNAKRSKPIFPAALLNISPNKTAMDKTKENILPEPKVRNIFGNRPANKRKNMFADFVVSESEDEIPELQPRVFGFQKKL

EQRRISSISKGREGSPASSITTDMEMDDWKLLPSSTMVENQLEDIVAGHTPKRARLSKLSEAKESEAGTLQTNTTGDKTKSSNKSIMSKN

KSKSSPDAKDKSLNSSRTTRSMLKNASKLEENTEPKQDTKTQSKISTKDNALDVSLSKATTKQNTSLNKSLRLNKSRTRNSSKLNEEEM

ETDENVVKNAINDATRVISKNASSAAVASKSINEKERTITLKVHDKASETGSNKEEDDNNFVLQYEDEIVEEATDHNQINENKTTKIKNRNTI

VIENDQETDVNESKSNKSIQKEPKQAKTVEESVESQNEQSGNDNIDGNEIEENAIEQNHESHDGEKISRELNEESNNSEGNDERNVTKDN

KDQENDEEINESQENDEEINLSQESNRDEAVLSRVDEENESEVNEESENEDDVNESNESEEQNESQEVEAEVDESEEIEEQNESQEIENE

ANESQEVNEEADDSKVEENASEDEEENQEVENDEEENAEESQEVDNEESQEIENQEEFEVSQESEHEVSQEVENEEENEEENDEGDQE

VEQEEENQSEDDVEDQVEDQSQEIENDEENDEEENEESQEIENDEENVVESEAEVNESQEIEGDQEMDDSDEADRAIASDPEISDQDDN

GADEMEVDDDDDNNEQESENEEETEGNQEESQEEQQEPSAEEIEESPNVTHDTTGRHRQKVLKSPEAILHDKTNQMDSFTAKGRNTS

IRKTKSMIKNLNIRPSLAPQRDSLAFSDGTRDSSAEGSGWDSHRTTRKTLRQTFGKDFTPRKSLRALVMEKSAKRQTEHMDLHSETSKY

PQANSTELAEDSNGHVDDFVESDHEVSRRTRQTTLETYLQKIKQKNLENKVKMEELVRNSLKAPARDTLSLFKVPNKPAPRRLKPTQNK

PRTQVKAITAFGELPTEVIEDMKYKPPKRFQPTNASWITKRLYKFLETKLEPKYDYKARVRAEKLVETIYHFAKDLRRHDVAPTDAVDVLK

HELARLEVVQTHFEFYEFFHEFMPREVRVKVVPDIVNKIPLPRHGVFSDILRGNNVQG

> S. frugiperda CENP-N

MPLEVFCVTALPEFGVRWRELSKAVRNARLPMQPASVARVLCRHVSKALTADEIEDIVARLRLKLVATQPRTWHVIRLSEKTTEEPVTLTM

RAVPGRITQALRKSKKTMRPEVQTVLLGDMLYLSIQLVSEHKSGSALYVATPPGEPVALVSSVNMVGLIKATVEGLGYKSYEIADLHGRDI

PSLLRINDRAWNTNADHLAEIPEYAPTPIITETGIDYTYKAYDENYVENILGPNPPKITDLTIKTSKSFFDRSRLDKNINITINIKTEDLAKSLKC

WVSKGAIAPTSDLIKIFHQIKSNKISYTREDD

To express the C terminus of S. frugiperda CENP-T a 166 amino acid C-terminal fragment that contains the HFD and CENP-T

extension was isolated to generate the S. frugiperda CENP-T-HFDextension-FLAG pIBV5 expression clone.

For LacO/LacI tethering assays, pVS1-LacO (Addgene RRID: Addgene_33143 [45]) was modified to insert the blasticidine resis-

tance cassette from pIBV5 into the BamH1 and Sac1 sites. To generate LacI-GFP pIBV5, the LacI-eGFP insert was amplified from

eGFP_N1_LacI pDEST (kind gift from Genevieve Almouzni lab, Nuclear Dynamics unit, UMR3664, Institut Curie, Paris, France [46])

and cloned into pENTR and pIBV5. To generateB.moriCENP-T-GFP-LacI pIZV5, the full-length coding sequence ofB.moriCENP-T

from B. mori CENP-T-FLAG pIZV5, lacI from pCMV-lacI (kind gift from Genevieve Almouzni lab, Agilent Cat#217450) and eGFP from

eGFP_N1_LacI pDEST were isolated by PCR and assembled into the shuttle vector pRS416 (kind gift from Heloise Muller, Nuclear

Dynamics unit, UMR3664, Institut Curie, Paris, France) using the yeast-based homologous recombination-based DNA assembly,

described in detail in elsewhere [47, 74]. Briefly, S. cerevisiae BY4742 strains were transformed with the linear DNA fragments

and linearized pRS416, colonies were picked to isolate the resulting recombined construct. The constructs were then transformed

and amplified in E. coli. The S. frugiperda CENP-T-GFP-LacI was isolated from this plasmid and cloned into pIZV5 (Invitrogen

Cat#V800001) using KpnI and XbaI. To generate the B. mori DN-CENP-T(201-1013)-GFP-LacI pIZV5, a truncated B. mori CENP-

T-GFP-LacI fragment (without the part coding for the first 200 amino acids was isolated PCR and cloned into pIZV5 using BamHI

and XhoI sites.

To generate theB. moriCENP-T-FLAG pIZV5 construct, the coding sequence of B. moriCENP-T was isolated from a BmN4 cDNA

library, fused to the 3xFLAG tag by PCR amplification and cloned into pIZV5 using BamHI and XhoI. To generate the B. moriCENP-T

RNAi-resistant clone (B. moriCENP-Tres-FLAG pIZV5), we ordered a Gblocks gene fragment (IDT) to recode an internal sequence of

B.moriCENP-T from amino acid 221 – 334. A 50 fragment ofB.moriCENP-T that contains the recoded part was then assembled with

the 30 fragment of B. mori CENP-T fused to a 3xFLAG into pRS416 using yeast-based homologous recombination and subsequently

cloned into the into the BamHI and XhoI sites of pIZV5. To generate the B. mori DN-CENP-Tres-FLAG pIZV5 and B. mori DC-CENP-

Tres-FLAG pIZV5, B. mori CENP-Tres-FLAG pIZV5 was used as a template to isolate 50 or 30 truncated fragments to clone those into

pIZV5 using BamHI and XhoI. The constructs expressed CENP-T without the first 200 or last 112 amino acids, respectively.

To generate protein antigens for antibody production, we purified protein fragment expressed in bacteria. To generate the

S. frugiperda SUMO-6xHis-CENP-T (1-122) pT7 and B. mori SUMO-6xHis-CENP-T (1-218) pT7 bacterial expression constructs

the N-terminal domains of S. frugiperda (1-222aa) and B. mori (1-218aa) CENP-T were cloned into the pT7-His-SUMO vector (gift

from Ahmed El-Marjou, recombinant protein platform, Institut Curie) which included N-terminal 6X His and SUMO tags using Gibson

Assembly [75].

e6 Current Biology 30, 561–572.e1–e10, February 24, 2020

To generate the B. mori 6xHis-Spc24(73-162)-Spc25(70-211) pRSF-DUET1(Sigma Cat#71341) bacterial expression constructs,

the globular domain of B. mori Spc24 (73-162aa) was cloned with an N-terminal 6X His-tag into the first cassette of the pRSF-

Duet1 vector using the BamHI and PstI restriction sites. For dual expression of Spc24 and Spc25, the globular domain of B. mori

Spc25 (70-211aa) was also cloned into the second cassette of the same pRSF-Duet1 vector using the Nde I and Xho I restriction

sites. To generate the B. mori 6xHis-Spc24(73-162)-Spc25(70-211) pFASTBac-Dual for the recombinant baculovirus generation

to evaluate the Spc24/25 antibody, the Spc24 and Spc25 fragments were cloned into the BamHI and PstI, and KpnI and XhoI sites

of pFASTBac-Dual, respectively.

To generate the B. mori 6xHis Dsn1(1-100) pRSF-DUET1 and S. frugiperda 6xHis Dsn1(1-99) pRSF-DUET1, N-terminal region of

B. mori (1-100aa) and S. frugiperda (1-99aa) Dsn1 cloning into pRSF-Duet1 with N-terminal 6X His-tag BamHI + PstI.

Construction of cell lines

Around 1-5 mg of plasmid DNAwas transfected into 106BmN4 or Sf-9 cells usingCellfectin II (GIBCOCat#10362100) according to the

manufactor’s instructions. For IF experiments cells were grown on coverslips before transfections. For the generation of stable poly-

clonal cell lines, antibiotics were added 48 hours after transfection (300 mg/ml Zeocin (GIBCO Cat#R25001) or 40 mg/ml Blasticidin

(GIBCO Cat#R21001)). Selection was continued until no viable untransfected cells were observed.

Protein expression and affinity purification

The E. coli Bl21DE30 plys pRare (Sigma Cat#71400) was transformed with bacterial expression vectors containing the recombinant

genes. Bacterial cultures (4 L) were initially grown at 37�C, 220 rpm and then induced with 0.5mM IPTG (Euromedex Cat#EU0008-A).

Following expression at 37�C for 4 h, cultures were centrifuged at 6000 xg for 15min to pellet the cells. Cell pellets were resuspended

inWash Buffer (20 mM Tris pH 8, 300mMNaCl, 10mMMgCl2, 25 mM imidazole). The resuspended pellets were incubated at 4�C on

a roller with the addition of one tablet cOmplete Protease Inhibitor Cocktail (Roche Cat#11697498001), Triton X-100 (1% final con-

centration), and lysozyme (1mg/mL final concentration). Themixtures were sonicated at 30%amplitude for 8min total (30 s pulses) in

a Branson Digital Sonifier SFX550 (Branson Ultrasonics Corp.). Cell lysates were centrifuged at 30 000 xg for 1 h at 4�C. The soluble

fraction was removed and passed through 0.45 mmfilter units. The lysates were loaded onto Protino Ni-TED 1000 columns (Machery-

Nagel Cat#745110.50) that were pre-equilibrated with Wash Buffer. The columns were washed with 2-3 column volumes of Wash

Buffer and proteins were eluted with the following buffer (20 mM Tris, 250 mM imidazole, 300 mM NaCl, pH 8.0). The SUMO tags

were cleaved from the CENP-T proteins by digesting the eluates at 4�C with SUMO-Protease (enzyme produced by Ahmed El Mar-

jou, recombinant protein facility, Institut Curie) (final concentration 0.6 mg/mL). The eluates were loaded and migrated in Bolt 4%–

12% Bis-Tris Plus denaturing gels (Invitrogen Cat#NW04120BOX). The correct-sized bands corresponding to the proteins without

the SUMO tag were excised from the gels and sent to Covalab (Villeurbanne, FR) for generation of antibodies in rabbits. For the gen-

eration of antibodies against CENP-T and Dsn1, a 1:1 mix of B. mori and S. frugiperda CENP-T or Dsn1 protein fragments were in-

jected into rabbits. For the generation of the antibody recognizing Spc24 and Spc25 proteins, the isolated B. mori Spc24 and Spc25

protein fragments were injected.

Validation of CENP-T and Dsn1 antibody specificity

For each IP experiment, one confluent flask of BmN4 cells was used. Immunoprecipitation was performed as described previously

[76] omitting the cross-linking step and with somemodifications. Briefly, cells were spun down and washed with PBS with cOmplete

Protease Inhibitor Cocktail (Roche Cat#11697498001). Cells were resuspended in 140 mL lysis buffer (1%SDS, 10 mMEDTA, 50mM

Tris-HCl (pH 8.1)) with protease inhibitors and incubated for 10 minutes at 4�C. 1350 mL IP dilution buffer (1% Triton X-100, 2 mM

EDTA, 150 mM NaCl, 20 mM Tris-HCl (pH 8.1)) with protease inhibitors and 4.5 mL CaCl2 (1 M) were added and samples were incu-

bated for 2 minutes at 37�C. Chromatin was digested using I UNIT of MNase (Sigma Cat#N3755-500UN) for 15 minutes at 37�C. The

reaction was stopped by adding 30 mL EDTA and 60 mL EGTA followed by mild shearing and solubilization step using the Covaris

E220 Evolution ultrasonicator (Covaris) (150 s, peak power 75, duty factor 10, cycles/burst 200). Samples were spun down for 5 mi-

nutes at 16000 xg and the soluble extract was added to 50 mL magnetic Dynabeads Protein A (Invitrogen Cat#10002D) covalently

conjugated to 5 mL of rabbit polyclonal anti-CENP-T (this paper) or anti-Dsn1 (this paper) immunoserum or 10 mg anti-FLAGM2 anti-

body (Sigma Cat#F1804; RRID: AB_262044) as a control. Immunoprecipitation was performed for 15 minutes at room temperature

and samples were washed three times in PBS. Samples were digested on beads for MS analyses (Figures S5A and S5B) (see below).

Validation of B. mori Spc24/25 antibody specificity

The 6xHis-Spc24(73-162)-Spc25(70-211) pFASTBac-Dual construct was used to generate recombinant baculovirus DNA. Briefly,

pFASTBac-Dual constructs were transformed into DH10bacLL strain to produce and isolate the recombinant baculovirus backbone.

Recombinant baculoviruses were amplified in Sf9 cells to the generate high-titer virus stocks of B. mori Spc24/Spc25 baculovirus.

500 ml of the recombinant Spc24/Spc25 expressing baculovirus stock (MOI 100) were used to infect 50ml Sf9 cells (106 cells/ml) for

3 days. To obtain total cell extracts, the cell pellet was resuspended in buffer A (20 mM Tris pH 8, 300 mM NaCl, 5% glycerol, one

tablet cOmplete Protease Inhibitor Cocktail (Roche Cat#11697498001) and 20ml DNase I (Roche Cat#04716728001)) and incubated

for 20 min at 4�C. The samples were sonicated at 20% amplitude for 3 min and 30 s total (30 s pulses) in a Branson Digital Sonifier

SFX550 (Branson Ultrasonics Corp.) and centrifugated for 30min at 8000 rpm, at 4�C. The supernatant was used for the subsequent

experiments. The lysates were loaded onto Protino Ni-TED 1000 columns (Machery-Nagel) that were pre-equilibrated with Wash

Current Biology 30, 561–572.e1–e10, February 24, 2020 e7

Buffer. After a 30 min incubation at 4�C, the columns were washed in Wash Buffer with 20 mM imidazole followed by several elution

steps with increasing concentrations of imidazole (50mM, 100mM, 200mMand 500mM). Proteins were separated on 4%–20%Tris

glycine gels (Invitrogen Cat#XP04200BOX) and visualized using InstantBlue (Sigma Cat#ISB1L).

For protein blot analysis, samples were separated on Bolt 4%–12% Bis-Tris Plus gels (Invitrogen Cat#NW04120BOX) and trans-

ferred to a PVDFmembrane (Bio-RadCat#170-4272) using the Trans-Blot Turbo Transfer System (1.3 A, 25 V, 10min). Themembrane

was blocked using the Odyssey Blocking buffer (LI-COR Cat#927-50000) before primary antibody incubation (rabbit polyclonal anti-

Spc24/Spc25 (this paper), 1:1000 dilution and mouse monoclonal anti-6xHis, 1:1000 dilution (Sigma Cat#ab18184; RRI-

D:AB_444306)) and secondary antibody incubation (IRDye 680RD Goat anti-Rabbit IgG (LI-COR Cat#926-68071; RRI-

D:AB_10956166), IRDye 800CW donkey anti-mouse IgG (LI-COR Cat#926-32212; RRID:AB_621847), dilution 1:10000). The signals

were visualized on an Odyssey LI-COR scanner (Figure S5C).

Affinity co-immunoprecipitations

Cultures of the following Sf9 strains expressing full length or partial S. frugiperda kinetochore proteins fused to a C-terminal or N-ter-

minal 3XFLAG tags or control wild-type Sf9 cells were grown to exponential phase in Sf-900 II SFMmedium (GIBCOCat#10902-088):

CENP-I, CENP-M, CENP-N, CENP-T, CENP-T-HFDextension, Dsn I and Nnf1. For each strain, 3 3 109 cells were harvested by

centrifuging for 10 min at 300 xg, and the pellets were washed twice in cold PBS. The pellets were resuspended in 5 mL HDG150

Buffer (20 mM HEPES pH 7.0, 150 mM KCl, 10% glycerol, 0.5 mM DTT, 1 tablet cOmplete Protease Inhibitor), and then cells

were disrupted with 50 strokes in a dounce homogenizer at 4�C. The dounced fraction was centrifuged at 1700 xg for 10 min at

4�C, and the nuclei (lower fraction) were gently resuspended with 5 mL HDG150 Buffer and re-centrifuged in the same conditions.

The nuclei were resuspended in a final volume of 5 mL using HDG150 Buffer. Nuclear extracts were prepared by passing the nuclear

fraction 10 times through a 20G 1 1/2’’ needle (0.93 38 mm) and then 5 times through a 25G 3/8’’ needle (0.53 16 mm). The nuclear

extracts were centrifuged at 20 000 xg for 10 min at 4�C in microcentrifuge tubes. The pellets were resuspended in HDG150 Buffer

and pooled together at a final volume of 5 mL. To prepare the chromatin, the nuclear extracts were digested with �40 units MNase

(Sigma Cat#N3755-500UN) for 1 hour at 4�C on a roller, 3 mM CaCl2 was added to the digestions. The MNase digestions were

stopped by adding 250 mL of 0.2 M EGTA. To solubilize the digested chromatin, 10 mL of HDG400 Buffer (20 mM HEPES pH 7.0,

400mMKCl, 10%glycerol, 1 mMDTT, 0.05%NP-40, 1 tablet cOmplete Protease Inhibitor) was added to the samples and incubated

for 2 h at 4�C on a roller. The samples were centrifuged at 8000 xg for 10 min at 4�C. The supernatants were saved to bind to the anti-

FLAG M2 beads (Sigma Cat#M8823; RRID:AB_2637089). The M2 magnetic beads were prepared according to the manufacturer’s

recommendations. The digested chromatin samples were incubated with 150 mL anti-FLAGM2 beads. The beads were washed four

times with 1 mL HDGN320 Buffer (20 mM HEPES pH 7.0, 320 mM KCl, 10% glycerol, 1 mM DTT, 0.05% NP-40, 1 tablet cOmplete

Protease Inhibitor). For proteomic analyses of full-length kinetochore protein IPs, beadswere boiled in sample buffer to first run those

into gels (see below). For proteomic analyses of the CENP-T-HFDextension IP, proteins were directly digested on beads (see below).

For the silver stainings of Sf9 kinetochore IP samples shown in Figure S1, M2 beads were incubated with FLAG peptide (Sigma

Cat#F4799) diluted to a final concentration of 150 mg/mL in 750 mL TBS Buffer (50 mM Tris-HCl, pH 7.4, with 150 mM NaCl) for

1 h at 4�C on a roller. The supernatants were removed and the eluates were concentrated in Amicon Ultra-0.5 mL 3K MWCO filters

(Sigma Cat#UFC5003) according to the manufacturer’s recommendations. Samples were loaded and migrated on Novex 16% Tris

Glycine Precast Gels (Invitrogen Cat#XP00162BOX). Silver stains were performed on the gels using the Pierce Silver Stain Kit

(Thermo Fisher Scientific Cat#24612) according to the manufacturer’s instructions. Several bands were excised for mass

spectrometry.

Proteomics and Mass Spectrometry Analysis

IP enriched proteins of full-length kinetochore protein IPs and control samples were separated on 10%NuPAGE 10%Bis-Tris protein

gel (Invitrogen Cat#NP0301BOX) and stained with colloidal blue (LabSafe GEL Blue GBiosciences Cat#786-35). SDS-PAGE was

used with short separation as a clean-up step, and 4 gel slices were excised. Gel slices were washed and proteins reduced with

10mMDTT (Euromedex, Cat#EU0006-B) prior to alkylation with 55mM iodoacetamide (SigmaCat#I6125). After washing and shrink-

ing the gel pieces with 100%MeCN (Merck Cat#1.00099), in-gel digestion was performed using trypsin/Lys-C (Promega Cat#V5071)

overnight in 25 mM NH4HCO3 (Fluka, Cat#09830) at 30�C. Peptides were then extracted using 60/35/5 MeCN/H2O/HCOOH (Fluka

Cat#94318) and vacuum concentrated to dryness. Protein on beads samples (CENP-T-HFDextension IP and CENP-T and Dsn1 anti-

body validations) werewashed twicewith 100 mL of 25mMNH4HCO3 and submitted to on-beads digestionwith 0.2 mg of trypsin/Lyc-

C for 1h. Digested sample were then loaded onto homemade C18 StageTips for desalting and peptides were eluted using 40/60

MeCN/H2O + 0.1% formic acid and vacuum concentrated to dryness. Gel samples were chromatographically separated using an

RSLCnano system (UltiMate 3000 RSLCnano, Thermo Fisher Scientific) coupled to an Orbitrap Fusion mass spectrometer (Q-OT-

qIT, Thermo Fisher Scientific with a Nanospay Flex ion source (Thermo Fisher Scientific) and bead samples were also analyzed

with a Q Exactive HF-X mass spectrometer. Peptides were first trapped on a C18 precolumn (300 mm inner diameter x 5 mm; Invi-

trogen Cat#160454) at 20 ml/min or 2.5 mL/min with buffer A (2/98 MeCN/H2O in 0.1% formic acid). After 3 min or 4 min of desalting,

the precolumn was switched on line with the analytical C18 column (75 mm inner diameter x 50 cm; nanoViper Acclaim PepMapTM

RSLC, 2 mm, 100A, Thermo Fisher Scientific Cat#164535) equilibrated in buffer A. Separation was then performed with a linear

gradient of 5% to 25% or 30% buffer B (100% MeCN in 0.1% formic acid) at a flow rate of 300 nL/min over 100 min or 91 min.

e8 Current Biology 30, 561–572.e1–e10, February 24, 2020

MS full scans were performed in the ultrahigh-field Orbitrap mass analyzer in ranges m/z 400–1500 or m/z 375-1500 with a resolution

of 120 000 at m/z 200, ions from each full scan were HCD fragmented and analyzed in the linear ion trap or orbitrap.

For identification, the data weremerged and searched against the S. frugiperda corn strain proteome [59] using SequestHF through

Proteome Discoverer (version 2.2, Thermo Fisher Scientific) with the S. frugiperda CENP-T, CENP-I, CENP-N, Nsl1 candidate and

Spc25 (which were all not correctly annotated in the S. frugiperda corn strain assembly) manually added to the proteome database.

For identification of proteins in the B. mori IPs, the data were searched against the B. mori proteome. Enzyme specificity was set to

trypsin and a maximum of two-missed cleavage sites were allowed. Oxidized methionine, Carbamidomethyl cysteines and N-termi-

nal acetylation were set as variable modifications. Maximum allowed mass deviation was set to 10 ppm for monoisotopic precursor

ions and 0.6 Da for MS/MS peaks. The resulting files were further processed using myProMS [56] v3.6. FDR calculation used Perco-

lator and was set to 1% at the peptide level for the whole study.

For the kinetochore IPs on full-length lepidopteran proteins identified proteins that were at least four-fold enriched over the control

(the two controls were combined) with a minimum of seven derived peptides were analyzed further (Data S1). For the S. frugiperda

CENP-T-HFD-FLAG IP (Figure S1B) proteins that were at least four-fold enriched over the control with at least 5 derived peptides

were selected for further analyses (see above). For Figure 1, known kinetochore homologs or proteins with at least seven derived

peptides and that were enriched at least four-fold in three kinetochore immunoprecipitates are listed.

Immunofluorescence

Cells were grown on glass coverslips and fixed with ice cold MeOH (anti-CENP-T and anti-Spc24/25), ice cold acetone (anti-Dsn1)

and 4% PFA (anti-tubulin), followed by permeabilization using 0.3% Triton X-100 in PBS and blocked in 3%BSA-PBS. The following

antibodies were used: rabbit polyclonal anti-CENP-T (this paper, rabbit 045), rabbit polyclonal anti-Spc24/25 (this paper, rabbit

1621016) and polyclonal rabbit anti-Dsn1 (this paper, rabbit 1615031) generated by Covalab (Villeurbanne, FR) at the dilution

1:1000, anti-a-tubulin monoclonal Alexa Fluor 488 (Thermo Fisher Scientific Cat#53-4502-80; RRID:AB_1210526) at 1:1000, anti-

FLAG M2 mouse monoclonal (Sigma Cat#F1804; RRID:AB_262044) at 1:1000, anti-phospho Histone H3-Ser10 rat monoclonal

(Sigma Cat#MABE939) at 1:1000. For fluorescent conjugated secondary antibodies, we used goat anti-rabbit IgG Alexa Fluor 568

(Thermo Fisher Scientific Cat#A-11011; RRID:AB_143157) at 1:1000, goat anti-rat IgG Alexa Fluor 568 (Thermo Fisher Scientific

Cat#A-11077; RRID:AB_2534121) at 1:1000, goat anti-rat IgG Alexa Fluor 488 (Thermo Fisher Scientific Cat#A-11006; RRI-

D:AB_2534074) at 1:1000, goat anti-mouse IgG Alexa Fluor 488 (Thermo Fisher Scientific Cat#A-11029; RRID:AB_2534088) at

1:1000, goat anti-mouse IgG Alexa Fluor 568 (Thermo Fisher Scientific Cat#A-11004; RRID:AB_2534072) at 1:1000 and goat anti-

rat IgG Alexa Fluor 633 (Thermo Fisher Scientific Cat#A-21094; RRID:AB_2535749) at 1:1000. DNA was stained with DAPI (Sigma

Cat#D9542) and samples were mounted in Vectashield Antifade Mounting Medium (Vector Laboratories Cat# H-1000;

RRID:AB_2336789).

For anti-tubulin staining cells were fixed three and five days after RNAi-mediated depletion using a protocol for the preservation of

the whole cytoskelon [77]. Cells were washed with PBS for 5 minutes, then incubated for 10 min at room temperature in 1 mM dithio-

bis(succinimidyl propionate, DSP) (Thermo Fisher Scientific Cat#22585) in Hank’s balanced salt solution (HBSS) (GIBCO

Cat#14025050), followed by an incubation for 10 min at room temperature in 1 mM DSP in microtubule-stabilizing buffer (MTSB).

Cells were next washed for 5 min in 0.5% Triton X-100 in MTSB and then fixed in 4% PFA in MTSB for 15 min at room temperature.

After a 5minwash in PBS, cells were incubated for 5min in 100mMglycine in PBS, thenwashed again in PBS for 5minutes and finally

nuclei were stained with DAPI (Sigma Cat#D9542) and samples were mounted in Vectashield Antifade Mounting Medium (Vector

Laboratories Cat# H-1000; RRID:AB_2336789).

Microscopy

Images were acquired on Zeiss Axiovert Z1 light microscope. Z sections were acquired at 0.2 mm steps using 100X 1.4 NA oil

objective.

Quantification of fluorescence intensity was performed using the Fiji software [57] on unprocessed TIFF images. Mitotic cells

(H3S10ph positive) were quantified. For RNAi depleted cells, we first annotated the cells using an automated system (kind gift

from Solene Herv�e, Fachinetti lab, UMR144, Institut Curie, Paris, France). H3S10ph signals were then used as markers to manually

select, using the freehand selections tool, the nuclear area. The mean fluorescence intensity of each nucleus wasmeasured and cor-

rected for background. For background correction, the average of mean intensities of three random circular regions of fixed size

(30x30 pixels) placed outside nuclear areas was determined.

To quantify the fluorescent signal intensities for LacO/LacI tethering assays, the mean fluorescence intensities of CENP-T, Dsn1

and Spc24/25 signals were first measured in circular regions of fixed size (10x10 pixels) overlapping GFP-LacI fusion protein foci

(visualized by the GFP signals). Then, themean fluorescence intensity of CENP-T, Dsn1 and Spc24/25 at endogenous loci was deter-

mined as the average in three random circular regions of fixed size (10x10 pixels) placed over themitotic chromosome. As before, the

background intensity was also measured as the average of mean fluorescence intensities of three random circular regions of fixed

size (10x10 pixels). Both the mean fluorescence intensities overlapping the GFP foci or the endogenous loci were corrected with this

background value. For the quantifications in Figure 4, the ratio between the mean fluorescence intensity over the GFP foci and over

endogenous loci was calculated and plotted. A ratio above 1 means that the respective kinetochore components are enriched over

GFP fusion protein foci and therefore indicates recruitment by the tethered protein. In contrast, a ratio around 1 or below indicate that

kinetochore components are not preferentially enriched or are even depleted over the GFP fusion protein foci compared to

Current Biology 30, 561–572.e1–e10, February 24, 2020 e9

endogenous loci. While the corrected values of the fluorescence intensities at the endogenous loci were always positive, the cor-

rected fluorescence intensities overlapping the GFP foci can be negative in cases where the immunosignal was low and even below

the average background intensity. Therefore, the ratio of (corrected immunosignal overlapping the GFP foci/ corrected intensities at

the endogenous loci) can be negative.

For statistical analysis the Mann-Whitney test (unpaired, non-parametric test) was used to compare two ranks, using GraphPad

Prism version 8.12 for Mac (GraphPad Software, https://www.graphpad.com/scientific-software/prism/). Differences were consid-

ered statistically significant at values of P values% 0.05.

RNAi-mediated knock-down

BmN4-SID1 cells were grown on coverslips and incubated with 400pg/ml dsRNA for three days. After three days, the medium was

change to add another 400pg/ml dsRNA. After three or five days, cells were fixed and processed for IF as described. Primers used to

generate the DNA templates fused to T7 promoter for dsRNA generation are listed in Table S5.

RNA blot analyses

Total RNA was isolated using TRIzol (Invitrogen Cat#15596018) following the manufactor’s instruction. RNA blots were performed

using around 10 mg total RNA (CENP-T, CENP-I, CENP-N, Nsl1, Mis12, Spc25) or polyA-selected mRNAs from 20 mg total RNA

(CENP-M, Spc24) per lane. RNA samples from cells incubated for five days with respective dsRNAs were treated with glyoxal using

NorthernMax-Gly Sample Loading Dye (Thermo Fisher Scientific Cat#AM8551). The samples were loaded on a 1% agarose gel pre-

pared using NorthernMax-Gly Gel Running Buffer (Thermo Fisher Scientific Cat#AM8678) according to the manufacturer’s instruc-

tions. The RNA was blotted onto a Nytran membrane in 20X SSC (175.3 g NaCl, 88.2 g sodium citrate in 1.0 l water adjusted to pH 7)

using the TurboBlotter System (Bio-Rad Cat#1704155). After UV crosslinking RNA to the membrane, glyoxal treatment was reversed

by incubating the membrane in 10 mM Tris-HCl pH 8 for 20 minutes at room temperature. The membrane was incubated in 12 mL

QuickHyb solution (Agilent Cat#201220) with 1.0 mg salmon-sperm ssDNA (Sigma Cat#31149) for 1 hour at 65�C. Body-labeled anti-

sense riboprobes against kinetochore mRNAs and the loading control were prepared by using PCR products as templates for in vitro

transcription (MAXIscript T7 Transcription kit, Thermo Fisher Scientific Cat#AM1312). A radiolabeled probe against B. mori Rpl32

mRNA served as loading control. After an overnight hybridization at 65�C with radio-labeled probes, the membrane was washed

twice in 2X SSC, 0.1% SDS for 5minutes and once in 0.2X SSC, 0.1% SDS for 30 minutes. The membranes were exposed to phos-

phoimaging plates and analyzed using a Typhoon TRIO Imager.

CRISPR-mediated genome editing in B. mori

The non-diapause strain N4maintained at the University of Tokyo was used. All larvae were fed with fresh mulberry leaves or artificial

diet SilkMate (NOSAN SilkMate PS) under a continuous cycle of 12-h light and 12-h darkness at 25�C. Unique single-guide RNA

(sgRNA) target sequences in the silkworm genome were selected using CRISPRdirect (https://crispr.dbcls.jp/) [58]. The sequence

specificity in N4 strain was also checked by SilkBase (http://silkbase.ab.a.u-tokyo.ac.jp) [61]. Primers used for sgRNA transcription

in vitro are listed in Table S5. The sgRNAwas transcribed in vitro according to a method reported previously [78]. A mixture of sgRNA

(400 ng/mL) and Cas9 Nuclease protein NLS (120 ng/mL; NIPPON GENE Cat#319-08641) in injection buffer (100 mM KOAc, 2 mM

Mg(OAc)52, 30mMHEPES-KOH; pH 7.4) was injected into each eggwithin 3 h after oviposition [79]. The injected embryos were incu-

bated at 25�C in a humidified Petri dish until hatching. Injected individuals were crossed with non-injected individuals to obtain G1

broods. To identify G1moths in whichmutant alleles were transmitted fromG0, genomic DNAwas extracted from aG1 adult leg using

the hot sodium hydroxide and Tris (HotSHOT) method [44]. Genomic PCR was performed using KOD OneTM (TOYOBO Cat# KMM-

101) under the following conditions: 35 cycles of denaturation at 98�C for 10 s, annealing at 60�C for 5 s, extension at 68�C for 5 s.

Primers used for mutation screening are listed in Table S5. The PCR products were denatured and reannealed at 95�C for 10 min,

followed by gradual cooling to 25�C. Mutations at the target site were detected by heteroduplex mobility assay using the MultiNA

microchip electrophoresis system (SHIMADZU) with the DNA-500 reagent kit (SHIMADZU Cat#292-27910-91) [80, 81]. A mutation

at the target site was sequenced using BigDye� Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems Cat#4337454) and ABI

PRISM� 3130xl Genetic Analyzer (Applied Biosystems). Wemaintained the mutant line and obtained eggs carrying the homozygous

mutation by crossing between two heterozygous mutants.

QUANTIFICATION AND STATISTICAL ANALYSIS

Statistical details of experiments are detailed in the Figure legends, including the number of biological replicates (n = number of cells),

the definition of center, dispersion and precisionmeasures (mean, SEM and SD). Statistical analyses were performed with GraphPad

Prism 8.12 for Mac.

DATA AND CODE AVAILABILITY

The mass spectrometry proteomic datasets generated during this study are available at the ProteomeXchange Consortium via the

PRIDE [82] partner repository with the dataset identifier PRIDE: PXD016092 (username: [email protected], password:

on7Kq9rP).

e10 Current Biology 30, 561–572.e1–e10, February 24, 2020

Current Biology, Volume 30

Supplemental Information

CenH3-Independent Kinetochore Assembly

in Lepidoptera Requires CCAN, Including CENP-T

Nuria Cortes-Silva, Jonathan Ulmer, Takashi Kiuchi, Emily Hsieh, GaetanCornilleau, Ilham Ladid, Florent Dingli, Damarys Loew, Susumu Katsuma, and Ines A.Drinnenberg

CENP-M

BAIT

CENP-I CENP-N CENP-T Dsn1 Nnf1 Control

10

15

25

35

55

70

100

130

180

kD

CENP-M

11797001

CENP-I

19290001

CENP-L

CENP-T

01205001

19785001

CENP-K-like

02906001

07362001

CENP-N

CENP-M

11797001

CENP-I

CENP-L

CENP-T

01205001

19785001

CENP-K-like

02906001

07362001

CENP-N

CENP-I

CENP-L

02906001

07362001

CENP-N

CENP-M

11797001

CENP-I

CENP-L

CENP-T

01205001

19785001

CENP-K-like

02906001

CENP-N

ATP syn ATP syn ATP syn

CENP-I

19290001

CENP-L

CENP-T

0120500102906001

07362001CENP-N

CENP-M

CENP-I

19290001

CENP-L

CENP-T

02906001

07362001Nuf2

Knl1 Knl1

Spc25

Nuf2

Ndc80 Ndc80

Dsn1

Nnf1

Nsl1-like

Mis12

Nsl1-like

Dsn1Mis12

Nnf1

Spc25

ATP syn

11797001

A

B

GSSPFG00032913001This studyGSSPFG00001205001 32

51 105.2150.051.3

CENP-TCoil-coiled RWD like protein

Phosphatidylinositol 3-kinase 3 isoform X1

MWG (kDa)

Number of peptides

in IPS. frugiperda ID Description

Coverage

(%)

30.4

44.7

GSSPFG00019785001GSSPFG00011797001

GSSPFG00011349001 13

1820 28.0

43.9

76.0

12.2

unknownCoil-coiled RWD like protein

CENP-I

Annotated as non-coding RNA

66.034.2

74.1

Number of peptides in control

316

0

0

00

GSSPFG00009519001GSSPFG00023246001 10

12 8.134.7 CENP-L

Uncharacterized protein47.224.5

GSSPFG00005844001 6 85.9 Atlastin 8.6

00

0

B. mori ID

KWMTBOMO14835KWMTBOMO06797KWMTBOMO14835

KWMTBOMO09290KWMTBOMO06154KWMTBOMO02221

LOC105842400

KWMTBOMO00944KWMTBOMO11447

KWMTBOMO03030

GSSPFG00013028001 5 129.9 Eukaryotic translation initiation factor 5B 7.4 0KWMTBOMO07723

Control (

n=2072)

LOC105842400

(n=454)

KWM

TBOM

O00944

(n=450)

0

10

20

30

Mit

oti

c In

de

x (%

)

dsR

NA

LO

C1

05

84

24

00

RNAi

dsR

NA

KW

MT

BO

MO

00

94

4 H3S10ph CENP-T

This study 28 36.4

48 12.1

GSSPFG00020888001 7 55.1 Transitional endoplasmic reticulum ATPase TER9424.5 1

GSSPFG00002981001 7 59.8 Uncharacterized protein1

KWMTBOMO11666

27.9KWMTBOMO06965

H3S10ph CENP-T

Figure S1: Composition of CenH3-deficient kinetochore in S. frugiperda. Related to Figure 1. A) Silver staining of SDS-PAGE separating protein complexes identified by immunoprecipitation using anti-FLAG affinity steps followed by elutions using the FLAG peptide from Sf9 cell lines stably expressing 3xFLAG-tagged inner kinetochore components (CENP-M, CENP-I, CENP-N and CENP-T) and outer kinetochore components (Dsn1 and Nnf1). Wild-type Sf9 cells (Control) were also used for affinity purifications with anti-FLAG antibodies. Kinetochore proteins used as baits are highlighted in red. The identity of several individual bands was determined by mass spectrometry (MS) analysis of digested peptides (blue). The theoretical size of other components identified in the IPs with at least 5 matching peptides are indicated. B) Absence of detectable lepidopteran CENP-W candidates in S. frugiperda FLAG-tagged CENP-T HFD and 2-helix extension (CENP-T-HFDexten-sion-FLAG) immunoprecipitates. Top: Table lists the number of peptides and coverages of S. frugiperda proteins identified by mass spectrometry enriched in the CENP-T-HFDextension-FLAG IP over the control. For MS analyses, samples were directly digested on beads. The corresponding homologs in B. mori and descriptions based on homology predictions are listed alongside. Two small proteins of unknown function are highlight-ed in green. Bottom: Depletion of B. mori homologs of the two small S. frugiperda proteins found in CENP-T-HFDextension-FLAG IPs had no detectable mitotic and CENP-T recruitment defect. Graph showing the percentage of mitotic cells (H3S10ph positive cells) three days after RNAi-mediated depletions of corresponding mRNAs (n= number of cells, ± standard error of the mean). Representative images of mitotic BmN4-SID1 cells showing the levels of endogenous CENP-T in cells upon depletion of two small B. mori proteins. Scale bar: 10μm. While we identified two small proteins with sizes similar to known CENP-W homologs in IP experiments pulling down S. frugiperda CENP-T C-terminus, RNAi-mediated depletions of B. mori homologs do not increase the number of mitotic cells or abolish CENP-T localization to mitotic chromo-somes. Both potential protein products (LOC105842400 is annotated as a non-coding RNA) have no detectable similarity to known CENP-W homologs.

B. mori CENP-T domain (P_KWMTBOMO06797)

2nd iteration E value 0.0025

Calypte anna (UniProtKB - A0A091IQR2)

1st iteration E value 3.0x10

-4

Homo sapiens (H3BTR4)

jackhmmer searches HHpred searches

B. mori CENP-T domain (P_KWMTBOMO06797)

Probability: 94.56 % E value: 0.055

Gallus gallus CENP-T (PDB 3B0D)

A

C

B

B. mori CENP-T (P_KWMTBOMO06797)

Probability: 75.4 % E value: 1.4

Gallus gallus CENP-T extension (PDB 3B0D)

B. moriS. frugiperdaH. melpomeneDanaus/1-106B. terrestrisA. melliferaA. cephalotesN. vitripennisA. pisum

R. prolixusF. auricularisL. vibransL. fulvaE. danicaA. domestica

P. humanus corporis

X. tropicalis

D. rerioH. sapiens

G. gallus

D. plexippus

2D structure

B. germanica

T T K R L Y K Y L - E D K L E P K Y - - - D Y K A R V R A E K L V E T I Y H F T K E V K K H E V A P N - - - - - - D A V D V L K H E M A R L D I V K T H F D F Y Q F F H D F M P R E I R V K V V P D I V - - - - - - N K I - - - T I P R N - G V F S E I L S G H A V H A

I T K R L Y K F L - E T K L E P K Y - - - D Y K A R V R A E K L V E T I Y H F A K D L R R H D V A P T - - - - - - D A V D V L K H E L A R L E V V Q T H F E F Y E F F H E F M P R E V R V K V V P D I V - - - - - - N K I - - - P L P R H - G V F S D I L R G N N V Q G

I T K R L Y K F L - E T K L E P K Y - - - D Y T A R V R A E K L V E T I Y N F T K N I K K Q N I A P S - - - - - - H S V D V L K N E M A R L N I V K T H F D F Y E F F N N Y M P R E I R I K V I P D I V - - - - - - N K I - - - P L P K H - G V F A D I R - - - - - - -

I T K R L Y K F L - E T K L E P K Y - - - D Y K A R I R A E K L V E C M Y T L T K E I R R H D T P R V - - - - - - D G V N E L K H E M A K L D L V K T H F D F Y E F C HQ F L P R E I R V K V V P D V V - - - - - - N K I - - - P L P K H - G V F S D I L R - - - - - -

I T D R L Y K Y L - WK C M E P R F - - - K L E T R V V S E K F I HQ L S S I T T L I A K R K S Y L N Y - - - - K A E L Y A L M K E M A R L D I I H T R N D F Y N F C H D F F P Y E L R V K T V P M L L P G N - - K R N I - - - P Y D A D - T L H K P L L V P - - - - -

I T D R L Y K Y L - WK HM E S R F - - - K L E T R L V S E K F I HQ L S N V T K F I M K C K S Y L D Y - - - - E N E L Y A L M K E M A R L G I I S T R N D F Y H F C Q D F F L Y E L R V K I V P M I L P G N - - K R N I - - - P Y D A N - K L N E P L L D S - - - - -

A T D R L Y K F L - WK R L E P K Y - - - K L A T R V K S E K F V Q E L A T V V A I I E R C K N Y E N Y - - - - N T E L K A L M K E M A R L K L I N T R MD F Y H F C Q D F L P Y E F R V K V I P M S L P G N - - E N N I - - - P F D P K - N V H T P L L D T E - - - -

V T D R L Y K Y L - WK HM E P K F - - - G L N T R V Q S E K F V M K L S N V F T V V T R R E M Y E N Y - - - - K E D V D D L M V S M A E L G I I K D R Y D F Y R F C R E Y M P Y S F R I K V V P MQ L P G N - - L R N I - - - P Y E P E - L V H K P I L P D R S G S Y

I T K R L Y K F L - I I K L Q P N Y - - - N I Y S V K Y A E K F V K Y L A Q S L K Q I L K D K V N L Q - - - - - - L Y T DM L R Y HMA R Y H I I K D T F D Y I G F L C D Y I P M E Y Y K K L V P GWN L - - - E T S A V - - - K F D P Q - K Y Y V P L M E D E E F - -

V T K K L HQ Y I - L N K L K P K Y - - - G I D T N V R A E E I E M R I C D T V K I V T R R K S Y Q K - - - - - - S L T E K L K K E M A T V G L I S T T V D F Y E - - - - - - - - - - - T K V I P C H H P - - - I T N S I Y - - P P K A A V N P F D E I L L K H - - - -

A T H R L Y K L I - I S T C E K K F G - - E I Q A Y K K A E K F V V W L C K Q V Q T V I R R K K L E N Y - - - - R D I A E S V K K A L Y R L G I I K S H H D Y F L F I E N Y L P F L Y R L K V T P C T R A Y N - R V S G I - - - P I P P G I G L F D D L K L - - - - - -

I K K E L Y T F L - E K K L G S K Y - - - S L E K R Y H S E R L V L L L H N A V T N T L L G H P E L S - - - - - - - - L Q S L K E E L I K Y N I C K T Q L D Y F T F L N L Y T P L E F I K K V I P M G R - - - - - - - - - - - - A Y S N E - D F K H E V I T V S C - - -

A S E R L Y K F L - Q S K C E S R R - - - - - - - - - K S E E I V L F L HQ T L K K L P Q T R P S D N Y D K R A L D L V E Q I K K K L Y K N S I I K T F WD L V I F F N E Y L P S E L S Q K A L P S V E F T E K G T R N I G - P P M T Q K - T I F E P L N K Y Y - - - -

A R H E L K T V V - I E T I E E Q E - - - - - D V L I D P N D F L L D L V D T I K L V M K S K K V K E V - - - - R Q G V Q K V K Q E L F K V R L I R T L G D F Y Q F V R D Y L P Y E F R R R S I P S S G Y H E - - F N P I Q - - E L E A D - - I D C P L F - - - - - - -

A R N E L K T V V - T E A I E E Q E - - - - - D C L L D P Q D F L F D L V D T I K L V M K S K R L K E V - - - - R Q G V Q K V K E E L F N L K L I R T L R D F Y Q F V R D Y L P Y E F R K R S I P S S G H Y K - - F N P V W - - E V E A D - - L D C S L F - - - - - - -

I T S K L C K K L - E T Q F A K K D - - - S L S A C R I T E D F I DW L S D K V T K A L K S K S D S H - - - - - - K M A R Q I A K T L K K H E F I E N D Y D F Y L F C N E Y L P Q A F C A K I F - - - - - - - - - - - - - - - - T Y P R E - K F I E - - - - - - - - - -

L H T R L Y K S L - S Q S V I M K L G K N V I N P E K E S E E I I S K L V S L I E K A L H Y R R K G C - - - - - N P F V D E V I K H L Y H S A I I K T P MN F M C F C M R F L P E R F I D K A L R D K D - - - - N D S G I L G K V I D R D L N L - - - - - - - - - - - -

S S S F V K Q L V - K H S T R M K V - - - S K D S Y K E V E T C L K V Y F E Q L C G D L T A Y AMH A N - - R K T I T C S D I E L L M R R Q G Y V T D AM P L N V L I E R H L P M E Y R R Y L I P C A S - - - - S G NQ V Y - - P K T K S - - - - - - - - - - - - - - -

G L S H Y V K L F - S F Y A K M P M - - - E R K A L E M V E K C L D K Y F Q H L C D D L E V F A A H A G - - R K T V K P E D L E L L M R R Q G L V T DQ V S L H V L V E R H L P L E Y R Q L L I P C A Y - - - - S G N S V F - - P A Q - - - - - - - - - - - - - - - - -

P K S Y V M S I F K K H F A K T K V - - - A S D V Y P V I N E I L K K Y F D R L A D D L E T Y A T H A K - - R K T I E V E D F E L L M R R Q G F V T D S M P V N V L I E K Y L P L E Y R K L L I P V A T - - - - S G N K V I - - P T Q R R - - - - - - - - - - - - - - -

A S S L I K Q I F - S H Y V K T P V - - - T R D A Y K I V E K C S E R Y F K Q I S S D L E A Y S Q H A G - - R K T V E M A D V E L L M R R Q G L V T D K M P L H V L V E R H L P L E Y R K L L I P I A V - - - - S G N K V I - - P C K - - - - - - - - - - - - - - - - -

2D structure

905

537

459810

665

α1 α2 α3

Histone fold domain CENP-T extension

799

12131148

1202

1982

11441248

1044

879

10452448

446

1516

54189

58

TAF9

CENP-S

H4

CEN

P-T

H2

AA

rch

aea

l his

ton

es

NZ

2NF2

H2B

H3 CenH3

CENP-X

CENP-W

Inse

ct C

EN

P-T

D

Figure S2: Prediction and evolution of insect CENP-T homologs. Related to Figure 1 and Figure 6. A) and B) Prediction of known

CENP-T homologs using the C-terminus of the B. mori CENP-T protein. Homology searches (arrows) and corresponding E values that link

the C-terminus of the B. mori CENP-T protein to other known CENP-T homologs including Calypte anna (Aves), the human CENP-T and

the G. gallus CENP-T domain structure indicated by species name and UniProt or PDB IDs. While the first jackhammer [S1] iteration only

picked up insect homologs, the best hits of the second iteration are known CENP-T homologs. The best hit of the HHpred [S2] search within

the PDB is the G. gallus CENP-T. The reciprocal best hit of a search using the G. gallus CENP-T HFD helix 3 and extension (amino acid

639-689) against the B. mori proteome was the B. mori CENP-T. B) Multiple alignment of the C-terminus of vertebrate and insect CENP-T proteins. B. mori (above) and G. gallus (below) secondary-structure predictions are derived from the Jpred4 (α-helical regions are in red; β-strands in yellow) [S3]. Background colouring of the residues is based on the clustalX colouring scheme. Full insect CENP-T sequences and accession numbers if available are listed in Table S1. C) Phylogenic distribution of known and insect CENP-T proteins as well as

various additional histone fold proteins. Maximum-likelihood phylogeny of HFD with known CENP-T proteins in orange and newly

identified insect CENP-T proteins in red. Bootstrap values above 80 are indicated by purple circles on the branches. The scale bar measures

the evolu-tionary distance in amino acid substitutions per site. The phylogenetic relationship between the insect proteins (red) and other

CENP-T proteins (orange) cannot be unambiguously resolved. Nevertheless, we refer to the insect proteins as CENP-T because of the

common sequence architecture with other CENP-T proteins; remote homology predictions identifying known CENP-T proteins as best hits outside of insects (Figure S2A and B) and the experimental evidence of kinetochore participation and outer kinetochore recruitment.

Notably, the branch at the root of the insect CENP-T proteins clade indicates a high degree of protein sequence divergence of the insect homologs, which could explain why their putative homology to other CENP-T homologs has been missed in previous searches.

CENP-T CENP-I CENP-N Nsl1 Mis12CENP-M Spc25

6000

4000

3000

2000

1500

1000

Spc24

KD CTRL KD KD KD KD KD KD KD

Rpl32CTRL CTRL CTRL CTRL CTRL CTRL CTRL

A

RNAi

Mis

12

Sp

c25

Nsl

1

B

RN

Ai

DNA Tubulin DNATubulin

Control (

n=2072)

CENP-T (n

=1896)

CENP-I (n

=1655)

CENP-M (n

=2296)

CENP-N (n

=1889)

Dsn1 (n

=1435)

Mis1

2 (n=1855)

Nsl1 (n

=1879)

Spc24 (n

=1557)

Spc25 (n

=1615)0

10

20

30

Mit

oti

c In

de

x (%

)

Cont

rol

CE

NP

-TC

EN

P-I

CE

NP

-MC

EN

P-N

Dsn

1M

is1

2S

pc2

4S

pc2

5

DNA Tubulin DNATubulin

Nsl

1

DNA Tubulin DNATubulin

C

D

EDNA Tubulin DNA Tubulin

F

Co

ntr

ol

CE

NP

-TC

EN

P-I

CE

NP

-MC

EN

P-N

DNA DNA

Dsn

1M

is1

2S

pc2

5S

pc2

4N

sl1

Day 3 Day 5R

NA

i

RN

Ai

Tubulin Tubulin

RN

Ai

Co

ntr

ol

CE

NP

-TC

EN

P-I

CE

NP

-MC

EN

P-N

Dsn

1M

is1

2S

pc2

5S

pc2

4N

sl1

RN

Ai

Figure S3: Depletion of kinetochore components affects mitotic progression in B. mori cells. Related to Figure 2. A) RNA blot analyses

showing mRNA levels encoding for various kinetochore components in control (CTRL - RNAi against GFP) and depleted cells (KD). The

star indicates the band of the respective mRNAs. A probe against Rpl32 mRNAs were used as loading control. The size marker is in base

pairs. An RNA probe targeting the Dsn1 transcript did not reveal any signal and is therefore not included in this figure. The efficient depletion

of Dsn1 protein is confirmed in our IF data analyses (Figure 3). We were unable to assign one band to the CENP-M transcript indicating the

possibility of multiple expression isoforms. The signal of all bands was decreased upon RNAi treatment indicating e¬fficient depletion of

CENP-M transcript levels. B) Quantification of the percentage of mitotic cells (H3S10ph positive cells) five days after RNAi-mediated

depletion of various kinetochore components (n= number of cells, ± standard error of the mean). C) Representative images of mitotic cells

stained with anti-tubulin used to classify mitotic defects observed at three days after depletion of outer kinetochore proteins Mis12, Nsl1 and

Spc25. Scale bar: 10μm. D) Representative images of mitotic cells stained with anti-tubulin with mitotic defects observed five days after depletion of inner and outer kinetochore components. Scale bar: 10μm. E) and F) Zoomed-out versions of representative images of cells stained with anti-tubulin used to classify mitotic defects observed three (E) and five (F) days after depletion of inner and outer kinetochore

components. Scale bar: 30μm.

DAPI Anti-FLAG DAPI Anti-FLAG

CE

NP

-T-F

LA

GC

EN

P-T

res-

FL

AG

C-C

EN

P-T

res-

FL

AG

ΔΔ

N-C

EN

P-T

res-

FL

AG

M e r g e M e r g e

A

B

3xFLAG CENP-T-FLAG (1037 aa)

Histone fold domain

and CENP-T extension

3xFLAG CENP-Tres-FLAG (1037 aa)

3xFLAGΔC-CENP-Tres-FLAG (925 aa)

3xFLAG ΔN-CENP-Tres-FLAG (838 aa)

133 aa

133 aa

133 aa

RNAi α CENP-TNo RNAi

Ad

de

d C

on

stru

cts

Figure S4: The B. mori CENP-T N- and HFD containing C-terminus are both essential for mitotic progression. Related to Figure 3. A)

Schematic representation of the wild-type FLAG-tagged CENP-T (CENP-T-FLAG), FLAG-tagged RNAi-resistant CENP-T

(CENP-Tres-FLAG), the N-terminal (ΔN-CENP-Tres-FLAG) and C-terminal truncated RNAi-resistant CENP-T (ΔC-CENP-Tres-FLAG). The

recoded region conferring RNAi-resistance to the used dsRNA against CENP-T is indicated in orange (see Methods). B) Representative IF images

of mitotic cells(n= 3 cells analyzed) expressing CENP-T-FLAG (first row) followed by RNAi-mediated depletion of endogenous and exogenous

CENP-T. Representative IF images of cells expressing full-length CENP-Tres-FLAG (second row), ΔN-CENP-Tres-FLAG (third row) or

ΔC-CENP-Tres-FLAG (fourth row) RNAi-resistant FLAG-tagged CENP-T followed by RNAi-mediated depletion of endogenous CENP-T. Scale

bar: 10μm.

P_KWMTBOMO06797

P_KWMTBOMO14073

P_KWMTBOMO05194

P_KWMTBOMO06603

P_KWMTBOMO14420 6

6

10

1057 116.6

81

171.2

104.4

106.6

CD2-associated protein

Acetyl-CoA carboxylase isoform X2

Uncharacterized protein LOC101742952

Heterogeneous nuclear ribonucleoprotein U-like protein 1

CENP-T

MWG (kDa)Number of peptidesB. mori ID Description

P_KWMTBOMO11045

P_KWMTBOMO11745

P_KWMTBOMO08735

P_KWMTBOMO08648

P_KWMTBOMO06463 7

11

13

1732 268.5

21.1

37.7

273.4

175.3

Dsn1

DNA repair protein RAD52 homolog

Spectrin alpha chain

DNA topoisomerase 2

Spectrin beta chain

MWG (kDa)Number of peptidesB. mori ID Description

A

B

C

P_KWMTBOMO14420 7 106.6 Heterogeneous nuclear ribonucleoprotein U-like protein 1

Coverage (%)

Coverage (%)

18.569.8

41.6

6.6

6.1

9.9

51.016.5

9.1

10.0

20.1

B. mori CENP-T (1013 aa)

Histone fold domain

and CENP-T extension

S. frugiperda CENP-T (1314 aa)

221 aa

218 aa

B. mori Dsn1 (179 aa)

S. frugiperda Dsn1 (117 aa)

99 aa

100 aa

B. mori Spc24 (162 aa)

B. mori Spc25 (211 aa)

142 aa

90 aa

Wash

(20 m

M)

Flow

-thro

ugh

E1 (50 m

M)

E2 (100 m

M)

E3 (200 m

M)

E4 (500 m

M)

Western Blot α-His Western Blot α-Spc24/Spc25Commassi

kDa

10

15

25

35

55

70

100

130

250

10

15

25

35

55

70

100

130

250

10

15

25

35

55

70

100

130

250

kDa kDa Wash

(20 m

M)

Flow

-thro

ugh

E1 (50 m

M)

E2 (100 m

M)

E3 (200 m

M)

E4 (500 m

M)

E1 (50 m

M)

E2 (100 m

M)

E3 (200 m

M)

E4 (500 m

M)

IP of His-Spc24 (72-162aa) - Spc25(69-142aa) from baculoviral-infected SF9 cells

His-Spc24 (12 kDa)

Spc25 (17 kDa)

H3S10P Test Protein

CENP-T

Dsn1

Spc24/Spc25

D E DAPI Plasmid CENP-T

CENP-T-GFP-LacI

ΔN-CENP-T-GFP-LacI

LacI-GFP

CENP-T-G

FP-LacI

ΔN-CENP-T

-GFP-L

acI

LacI-G

FP

-10000

0

10000

20000

30000

40000 ********

CE

NP

-T M

ean

Inte

nsi

ty (

A.U

)

Figure S5: Validating the specificity of antibodies raised against CENP-T, Dsn1 and Spc24/25 complex. Related to Figure 3 and

Figure 4. A) and B) Top: Schematics showing protein fragments used for antibody generation against lepidopteran CENP-T and Dsn1

homologs. Bottom: Mass spectrometry results identifying proteins (> 5 peptides) present in immunoprecipitates from B. mori soluble

extracts using the antibody raised against the CENP-T N-terminus or Dsn1, respectively, but not in immunoprecipitates using the Sigma M2

antibody as a control. C) Top: Schematics showing protein fragments used for antibody generation against the B. mori Spc24/25 complex.

Bottom: Coomassie staining and western blot analyses of different IP fractions pulling down B. mori His-Spc24/Spc25 protein fragments

expressed in Sf9 cells over Nickel-NTA columns. Two bands of the expected size for the His-Spc24 and Spc25 fragments are labelled with

a star on the coomassie-stained gel. Increased concentrations of immidazol are indicated in mM for the wash and each elution step. The α-His tag immunosignal confirms the presence of the His-Spc24 fragment in several elution fractions. A band of the same size in the same elution

fractions is also recognized by the custom-made antibody against B. mori Spc24/Spc25 fragments. An additional, much fainter, band corre-

sponding to the size of the B. mori Spc25 fragment is also detected. D) Immunofluorescence signals for CENP-T, Dsn1 and Spc24/Spc25 in

combination with histone 3 serine 10 phosphorylation (H3S10p) in mitotic BmN4 cells. Images from this panel are the same as in Figure 3.

White bar represents the scale of 10 μm. E) Immunofluorescence signals of CENP-T in BmN4 cells transfected with B. mori

CENP-T-GFP-LacI, ΔN-CENP-T-GFP-LacI or LacI-GFP fusion constructs. The anti-CENP-T immunosignal detecting endogenous CENP-T

in interphasic BmN4 cells is low or undetected probably due to the holocentric architecture. In cells transfected with full-length B. mori

CENP-T-GFP-LacI, however anti-CENP-T immunosignal are elevated due to the antibody recognizing ectopically expressed CENP-T

fusion protein. In contrast, in cells transfected with the B. mori ΔN-CENP-T-GFP-LacI, anti-CENP-T immunosignals are similar to those in

control cells expressing LacI-GFP indicating that the N-terminally truncated CENP-T fusion protein is not recognized by our CENP-T

antibody.

A

dsR

NA

Nsl

1

dsR

NA

Sp

c25

dsR

NA

Mis

12

H3S10ph Test Protein

CENP-T

Dsn1

Spc24/25

CENP-T

Dsn1

Spc24/25

H3S10ph Test Protein H3S10ph Test Protein

B

CE

NP

-T M

ean

Inte

nsi

ty (A

.U)

anti-CENP-T signal in RNAi treated cells

Sp

c24

/25

Me

an In

ten

sity

(A.U

)

anti-Spc24/25 signal in RNAi treated cells

Dsn

1 M

ean

Inte

nsi

ty (A

.U)

anti-Dsn1 signal in RNAi treated cells

CENP-T

Dsn1

Spc24/25

RNAi α G

FP

RNAi α M

is12

RNAi α N

sl1

RNAi α S

pc25

-2000

-1000

0

1000

2000

3000

4000***

*******

RNAi α G

FP

RNAi α M

is12

RNAi α N

sl1

RNAi α S

pc25

-2000

-1000

0

1000

2000

3000****

********

RNAi α G

FP

RNAi α M

is12

RNAi α N

sl1

RNAi α S

pc25

-500

0

500

1000 ***

****

Figure S6: Depletion of additional outer kinetochore components abolishes recruitment of their respective complex members in B. mori

mitotic cells. Related to Figure 3. A) Representative images of mitotic BmN4-SID1 cells showing the levels of endogenous CENP-T, Dsn1 and

Spc24/25 with and without depletion of outer kinetochore components (Mis12, Nsl1 and Spc25). Scale bar: 10 μm. B) Quantification of mean

fluorescence intensity for anti-CENP-T, anti-Dsn1 and anti-Spc24/25 signals upon kinetochore depletion compared to the control. Statistical

significance was tested using the Mann and hitney test (p . 1 for four stars, p . 1 for three stars, p . 1 for two stars and p . for one star).

♀ ♂

Table S2. Hatchability of the CENP-T mutant strain. Related to STAR Methods and Figure 5.

♀ ♂

Table S3. Genotypes of hatched larvae from the cross between two heterozygous CENP-T mutants. Related to STAR Methods and Figure 5.

Plasmid Identifier Description

143 S. frugiperda CENP-M-FLAG pIBV5

144 S. frugiperda FLAG-CENP-I pIBV5

145 S. frugiperda CENP-N-FLAG pIBV5

142 S. frugiperda CENP-T-FLAG pIBV5

146 S. frugiperda Dsn1-FLAG pIBV5

147 S. frugiperda Nnf1-FLAG pIBV5

150 S. frugiperda CENP-T-HFDextension-FLAG pIBV5

96 B. mori CENP-T-FLAG pIZV5

44 B. mori CENP-Tres-FLAG pIZV5

128 B. mori DC-CENP-Tres-FLAG pIZV5

81 B. mori DN-CENP-Tres-FLAG pIZV5

121 B. mori CENP-T-GFP-LacI pIZV5

157 B. mori DN-CENP-T-GFP-LacI pIZV5

149 pVS1-LacO-Blasticidin

99 LacI-GFP pIBV5

b027 S. frugiperda SUMO-6xHis-CENP-T (1-221) pT7

b030 B. mori SUMO-6xHis-CENP-T (1-218) pT7

b043 B. mori 6xHis-Spc24(73-162)-Spc25(70-211)

pRSF-DUET1

b036 S. frugiperda 6xHis Dsn1(1-99) pRSF-DUET1

b035 B. mori 6xHis Dsn1(1-100) pRSF-DUET1

b109 B. mori 6xHis-Spc24(73-162)-Spc25(70-211)

pFASTBac-Dual

Table S4. Plasmids generated in this study. Related to STAR Methods.

Oligonucleotide Source Identifier

DNA templates for dsRNA generation by T7 in vitro transcription

T7 GFP forward primer: TAATACGACTCACTATAGGGAGAGATGCCACCTACGGCAAG

This study GFP_T7_for

T7 GFP reverse primer : TAATACGACTCACTATAGGGAGACGCGGGTCTTGTAGTTGC

This study GFP_T7_rev

T7 B. mori Cenp-M forward primer : TAATACGACTCACTATAGGGAGATGAATGTTGAAGTAATCGAAAAGG

This study BomCenpM_T7_for

T7 B. mori Cenp-M reverse primer : TAATACGACTCACTATAGGGAGATTGCCATAGCATTCACAGGT

This study BomCenpM_T7_rev

T7 B. mori Cenp-I forward primer : TAATACGACTCACTATAGGGAGACAGTGGATTATGCTATTCAGTGG

This study BomCenpI_T7_for

T7 B. mori Cenp-I reverse primer : TAATACGACTCACTATAGGGAGAATTTTCCGGGACACACTCAG

This study BomCenpI_T7_rev

T7 B. mori Cenp-N forward primer : TAATACGACTCACTATAGGGAGAGCCTCTTCAACAATGCTGTG

This study BomCenpN_T7_for

T7 B. mori Cenp-N reverse primer : TAATACGACTCACTATAGGGAGAAGAGCATCGACACAGGCTTT

This study BomCenpN_T7_rev

T7 B. mori Cenp-T forward primer : TAATACGACTCACTATAGGGAGAGATCCTCCACAAAACCAACC

This study BomCenpT_T7_for

T7 B. mori Cenp-T reverse primer : TAATACGACTCACTATAGGGAGATTCTCGCATACCATTTCGTG

This study BomCenpT_T7_rev

T7 B. mori Mis12 forward primer : TAATACGACTCACTATAGGGAGAGGGAACGGATGAGGAATATG

This study BomMis12_T7_for

T7 B. mori Mis12 reverse primer :TAATACGACTCACTATAGGGAGAAGCAATGCAACTTCGTCTTTT

This study BomMis12_T7_rev

T7 B. mori Nsl1 forward primer :TAATACGACTCACTATAGGGAGAGGAGACGAAATGCGAGAATC

This study BomNsl1_T7_for

T7 B. mori Nsl1 reverse primer : TAATACGACTCACTATAGGGAGACAGTTTGCCGCCAATTTTAT

This study BomNsl1_T7_rev

T7 B. mori Dsn1 forward primer :TAATACGACTCACTATAGGGAGACCATCAGTGAAAATGAAATACAACA

This study BomDsn1_T7_for

T7 B. mori Dsn1 reverse primer :TAATACGACTCACTATAGGGAGACAAGTGCCATAACTTCTTTGACA

This study BomDsn1_T7_rev

T7 B. mori Spc24 forward primer :TAATACGACTCACTATAGGGAGAAGATTGGTGTGCCGTGCTAATT

This study BomSpc24_T7_for

T7 B. mori Spc24 reverse primer :TAATACGACTCACTATAGGGAGAGTCGGCAGAGTCCACTTCGAAA

This study BomSpc24_T7_rev

T7 B. mori Spc25 forward primer :TAATACGACTCACTATAGGGAGACTCATGAAGCCTATTTGCTAACT

This study BomSpc25_T7_for

T7 B. mori Spc25 reverse primer : TAATACGACTCACTATAGGGAGATACTTTATTTTGTTTAATGTTGAGAAA

CRISPR sgRNA transcription in vitro forward primer : GAAATTAATACGACTCACTATAGATGAACCAGAAAACAGTGCGTTTTAGA

GCTAGAAATAGC

This study N/A

CRISPR sgRNA transcription in vitro forward primer : AAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTT

ATTTTAACTTGCTATTTCTAGCTCTAAAAC

This study N/A

Mutation screening forward primer :ATCCAACGCAAGTAATGACGAT

This study N/A

Table S5. Oligonucleotides used in this study. Related to STAR Methods.

Oligos for CRISPR gene editing and screening:

Mutation screening reverse primer :ATCAGTGTTCGGGCTATTATCA

This study N/A

This study BomSpc25_T7_rev

Supplemental References

S1. Potter, S.C., Luciani, A., Eddy, S.R., Park, Y., Lopez, R., and Finn, R.D. (2018). HMMER web server: 2018 update. Nucleic Acids Res. 46, W200–W204.

S2. Zimmermann, L., Stephens, A., Nam, S.-Z., Rau, D., Kübler, J., Lozajic, M., Gabler, F., Söding, J., Lupas, A.N., and Alva, V. (2018). A Completely Reimplemented MPI Bioinformatics Toolkit with a New HHpred Server at its Core. J. Mol. Biol. 430, 2237–2243.

S3. Drozdetskiy, A., Cole, C., Procter, J., and Barton, G.J. (2015). JPred4: a protein secondary structure prediction server. Nucleic Acids Res. 43, W389-394.