Mitosis in diatoms: rediscovering an old model for cell division

Review article DOI 10.1002/bies.200900007

Mitosis in diatoms: rediscoveringan old model for cell divisionAlessandra De Martino,1 Alberto Amato,1 and Chris Bowler1,2*
1 Laboratory of ‘Molecular Biology of Photosynthetic Organisms’, UMR 8186, Department of Biology, Ecole Normale Superieure,46 rue d’Ulm, 75230 Paris cedex 05, France
2Stazione Zoologica Anton Dohrn, Villa Comunale, I-80121 Naples, Italy

Diatoms are important protists that generate one fifth ofthe oxygen produced annually on earth. These aquaticorganisms likely derived from a secondary endosymbio-sis event, and they display peculiar genomic and struc-tural features that reflect their chimeric origin. Diatomswere one of the first models of cell division and theseearly studies revealed a range of interesting featuresincluding a unique acentriolar microtubule-organisingcentre. Unfortunately, almost nothing is known at themolecular level, in contrast to the advances in otherexperimental organisms. Recently the full genomesequences of two diatoms have been annotated andmolecular tools have been developed. These resourcesoffer new possibilities to re-investigate the mechanismsof cell division in diatoms by recruiting informationfrom more intensively studied organisms. A renaissanceof the topic is further justified by the current interestin diatoms as a source of biofuels and for understandingmassive diatom proliferation events in response toenvironmental stimuli.

Keywords: cell division; diatom; mitosis; MTOC; spindle

Introduction

Our understanding of cell division derives almost entirely from

intensively studied model organisms. But the living world is

wider and more diversified than these elite models and many

organismal groups hide peculiarities that deserve to be

studied and understood. Diatoms (class Bacillariophyceae)

are one of the most species-rich algal groups and play a

fundamental role in marine primary production and in

biogeochemical cycles of several elements such as carbon,

silicon, nitrogen and phosphorus.(1) They derive from a

Abbreviations: Bb, basal body; CDK, cyclin-dependent kinase; EB1, end-

binding protein 1; EM, electron microscopy; GCP, g-tubulin complex protein; g-

tub, g-tubulin; KRP, kinesin-related protein; MC, microtubule centre; MT,

microtubule; MTOC, microtubule-organising centre; NE, nuclear envelope;

NEBD, NE breakdown; PC, polar complex; SPB, spindle polar body; TEM,

transmission electron microscopy; YFP, yellow fluorescent protein.

*Correspondence to: C. Bowler, Laboratory of ‘Molecular Biology of

Photosynthetic Organisms’, UMR 8186, Department of Biology, Ecole

Normale Superieure, 46 rue d’Ulm, 75230 Paris cedex 05, France.

E-mail: [email protected]

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secondary endosymbiosis in which a photosynthetic

eukaryote was engulfed by another eukaryote.(2) The guest

and host genomes then merged yielding a chimeric

genome.(3–5)

The most well-known feature of diatoms is their highly

ornamented external cell wall made of amorphous silica,(6)

called the frustule, which is composed of two unequal

valves, the epivalve fitting onto the hypovalve like a lid fits

onto its box. They predominantly divide vegetatively, through

mitosis and cytokinesis. During cytokinesis each daughter

cell inherits one maternal valve as the epivalve, while

the hypovalve is synthesised de novo within the mother

cell.(7,8)

Nuclear division and cytokinesis were studied in diatoms

as early as 1896 by Lauterborn(9) in an extensive dissertation,

which was then translated and summarised almost one

century later.(10) For several diatoms, mitosis has been

studied by light and transmission electron microscopy (TEM),

but there are only a few reports from a molecular standpoint.

The diatom genera for which mitosis has been most

commonly studied belong to the order Pennales (pennate

diatoms(7)), in particular Surirella (Supporting Information

Table S1). Many of these studies were innovative at that time

and contained impressive microscopic observations, and they

revealed a unique microtubule (MT)-organising centre

(MTOC), called the MT centre (MC). The MC shows peculiar

structure and behaviour during the cell cycle.

In the late 1980s, Cande and coworkers worked intensively

on spindle elongation in the centric diatom Stephanopyxis

turris both in vivo and by isolating mitotic spindles and

studying them in vitro using different approaches such as

indirect immunofluorescence.(11–13) Only in the mid 1990s

diatom mitosis was investigated molecularly, first in the

pennate Cylindrotheca fusiformis, revealing the role of

diatom spindle kinesin 1, a novel kinesin-related protein

(KRP), in spindle elongation.(14–16) Now that whole genome

sequences from diatoms are available(3,5) as well as high-

throughput reverse genetics,(17–19) the molecular mechan-

isms involved in diatom mitosis and the distinctive features

characterising diatom biology (see below and(7)) warrant

re-investigation.

BioEssays 31:874–884, � 2009 Wiley Periodicals, Inc.

A. De Martino, A. Amato and C. Bowler Review article

The microtubule centre of diatoms: anacentriolar MTOC with atypical featuresand behaviour during the cell cycle

The term MTOC was first defined by Pickett-Heaps(20) to

designate structures from which MTs emanate. MTOCs play a

crucial role in the division of eukaryotic cells. They nucleate

MTs and determine temporal and spatial distributions of the

interphase cytoplasmic MT arrays and the mitotic bipolar

spindle. Alterations of MTOC function lead to defects in

polarity, cytoplasmic organisation and fidelity of transmission

of genetic material during cell division. For instance, failure to

correctly regulate MTOC number or structure has dramatic

consequences in animal cells, leading to monopolar or

multipolar spindles, a characteristic of cancer cells.(21)

The MTOCs of yeast and slime mould look very different

from animal centrosomes (Fig. 1A–D) in that they are

multilayer structures lacking centrioles. In Saccharomyces

cerevisiae, the spindle polar body (SPB) is totally embedded

in the nuclear envelope (NE) throughout the cell cycle, and is

able to nucleate both nuclear and cytoplasmic MTs.(22) By

contrast, the SPB of the fission yeast Schizosaccharomyces

pombe(23) and the MTOC of Dictyostelium discoideum are

cytoplasmic, lying adjacent to the NE (Fig. 1A–D).

Centrioles likely derived from a basal body (Bb) that

nucleated MTs in flagella and cilia. It is thought that Bbs were

present in an ancestor, and during the evolution of eukaryotic

cells both structures (Bb and centrioles) were either

maintained or differentially lost, as in somatic cells of higher

plants.(24) Notably, angiosperms lack conspicuous MTOCs(25)

and MTs appear to be nucleated from several dispersed sites

organised as cortical parallel arrays encircling the inner

plasma membrane during interphase.

Bbs are still present in flagellated unicellular algae (e.g.

Chlamydomonas reinhardtii) and flagellated gametes of some

other algae (e.g. brown algae or centric diatoms), but have

been lost in pennate diatoms.(7) Centrics genetically differ

from pennates as much as humans from fish,(5) and such a big

distance is also reflected in their sexual cycle. Bbs in the

microgametes of centric diatoms are the only centriole-like

structures present among diatoms.

The diatom acentriolar MC has an ultrastructure different

from other MTOCs (Fig. 1). It diverges from those of animals

by the absence of centrioles and it diverges from yeast SBP

and slime mould MTOC because it has a spherical structure. It

also differs from the MTOC in brown algae that have an

animal-like centrosome consisting of a pair of centrioles and a

pericentriolar matrix.(26) This highlights the diversity of MTOC

structure and organisation even in phylogenetically closely

related organisms.

The first light microscopic descriptions of diatom MCs were

reported by Lauterborn at the end of the 19th century in the

pennate Surirella calcarata,(9) and this work represents one of


the first illustrations of mitosis and cytokinesis in a single living

cell. Later, TEM studies (Fig. 1, Supporting Information Table

S1) confirmed Lauterborn’s hypotheses.(9) The MC is clearly

visible by light microscopy in large pennate diatoms (several

species of the genera Surirella, Pinnularia and Nitzschia) and

in a few centric diatoms where it is said to be ‘less obvious’ to

observe (Supporting Information Table S1). It is always

closely associated with the interphase nucleus and with the

Golgi apparatus. In the centric diatom Lythodesmium the Bb is

involved in flagellum formation during sexual reproduction and

forms de novo during meiosis. For these reasons this

structure has been termed ‘paracentrosome’.(27) Centrics

may therefore produce Bbs de novo during flagellar formation

during sexual reproduction as in brown algae,(26) whereas in

pennates the centriolar structure has been totally lost.(28)

Besides its peculiar acentriolar ultrastructure, the diatom MC

also displays unusual behaviour during the cell cycle (Fig. 2

and 3). Animal centrosomes, yeast SPB and slime mould

MTOC are all present as single entities in the cell and they

duplicate before mitosis to constitute the spindle poles(29)

(Fig. 2). By contrast, the diatom MC (Fig. 2A) does not duplicate

before mitosis; rather a second structure, the polar complex

(PC), appears in premitotic cells (at early prophase in several

pennate diatoms) close to the MC. The PC is a cubic laminar

structure. At prophase, the PC splits into two parallel structures

known as the polar plates, which constitute the poles of the

mitotic spindle.(30) Both the MC and PC function as MT

nucleation centres but they act at different periods of the cell

cycle. Once the PC appears, the MC disintegrates and vice

versa. Lauterborn(9) already observed these as two indepen-

dent structures. The PC has been identified in several pennates

but the behaviour of MC/PC shows some variation among

diatoms (Supporting Information Table S1). It is thought that the

PC is produced by the MC(9,10) or forms very close to it,(31) but

almost nothing is known about their origin or whether the PC

derives from the MC. Ultrastructurally, the PC clearly differs from

the two centriolar poles of the mitotic spindle in animals

(Fig. 2B), while it is more similar to acentriolar yeast SBP

(Fig. 2C–D) and D. discoideum MTOC(22,32) (Fig. 2E). Further-

more, the duplication and splitting process of the PC recalls the

duplication and peeling off of the D. discoideum MTOC

(Fig. 2E). Again, the appearance of diatom MTOCs during

mitosis is very different from what is observed in higher plants,

which are characterised by broad spindle poles (not shown).

To conclude, the diatom MC is an MTOC model known for

many years with unusual and uncharacterised features that

merits being explored at the molecular level. The interconver-

sion of two spatially and temporally distinct structures during

the cell cycle is unique to the diatom MTOC, and the question

of whether the PC derives from MC components has not been

addressed at the molecular level. Why are there two spatially

distinct structures in diatoms, instead of a single entity that

duplicates as in the centrosome or SPB? An additional

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Figure 1. Diatom MTOC ultrastructure in an interphase cell compared to other MTOC models. A: Electron micrograph of animal centrosome

(left) and schematic representation of the pair of centrioles surrounded by a pericentriolar matrix (PCM) (right).(29) Reprinted with permission from

� Pearson Education, Inc., publishing as Benjamin Cummings (Campbell N. A. and Reece J. B., Biology, � 2002). B–E: Electron micrographs of

acentriolar MTOC of yeast (SPB) (B and C), slime mould (D) and pennate diatom (E). The SPB in S. cerevisiae (B) is a disc-like structure

embedded in the NE connected to cytoplasmic MTs (cMTs) and nuclear MTs (nMTs). The half bridge (HB) is the site of the new SPB assembly.(22)

Reprinted with permission from the Annual Review of Cell and Developmental Biology, Volume 20, � 2004 by Annual Reviews, www.

annualreviews.org. The SPB of S. pombe (C) is a single dense layer beside the NE (arrow).(23) Reproduced with permission from Ding

et al., 1997, Mol Biol Cell, 8 1461–1479, � The Biochemical Society. D. discoideum MTOC (D) is a matchbox-shaped three-layered structure.(110)

Reprinted with permission from the Molecular Biology of the Cell, Volume 10, �1999 by the American Society of Cell Biology. Pennate diatom

(Surirella ovalis) MC (E) is an extranuclear structure, appearing as a well-defined dark granule from which MTs radiate around the interphase

nucleus (n) and in all directions.(30) Reprinted with permission from Rockefeller University Press, � Tippit and Pickett-Heaps, 1977. Originally

published in The Journal of Cell Biology. 73: 705–727.

Review article A. De Martino, A. Amato and C. Bowler

question emerges from old electron microscopy (EM) studies:

does the MC in centric and pennate diatoms differ in

molecular structure?

Now that we benefit from two completed diatom gen-

omes,(3,5) molecular tools for reverse genetics,(17–19) and

time-lapse imaging of single cells overexpressing fluorescent

fusion proteins (paper in preparation), these questions can be

further re-examined. The centric (Thalassiosira pseudonana)

and pennate diatom (Phaeodactylum tricornutum) sequences

permit comparative studies to address the differences

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between the two diatom groups. Moreover, the existence of

distinct MTOC structures acting at different moments of the

cell cycle offers several advantages for understanding the

dual function of MTOC, i.e. orchestration of cytoplasmic MTs

in interphase and mitotic spindle organisation. Furthermore,

since MTOCs are integrative structures for several regulatory

pathways during the cell cycle, e.g. cyclin/cyclin-dependent

kinases (CDKs) and other regulatory proteins control

centrosome and SPB duplication in animal cells, it will be

interesting to explore the function of these components in


Figure 2. The MC/PC cycle in diatoms compared to MTOC duplication in animals, yeasts and slime moulds. A: Electron micrographs (upper

panels; reprinted with permission from Rockefeller University Press, � Tippit and Pickett-Heaps, 1977; originally published in The Journal of Cell

Biology 73: 705–727) and schematic representation (lower panels) of the MC/PC cycle and spindle development in the diatom Surirella. At

preprophase, the PC appears close to, but separate from, the interphase MC and elongates. The central spindle arises before prophase from the

PC that splits into two and gives rise to the ‘polar plates’ (PP). A band of densely packed, parallel MTs, the central spindle, develops between the

two PP. Condensed chromosomes become visible, distributed as a ring around the spindle at metaphase. Red circles and rectangles, MC and PC,

respectively; green lines, MTs; light green full lines and dotted lines, NE and partially broken down NE, respectively; blue circles, chromosomes.

B: In animal cells, centrioles disengage at early G1 phase and duplication of each centriole occurs at S phase. Daughter centrioles reach

maturation at G2. Following NEBD at prometaphase, the two pairs of centrioles become the focused spindle poles at metaphase. Red cylinders,

centrioles; in grey, pericentriolar matrix (PCM); green lines, MTs; light green full lines and dotted lines, NE and broken down NE, respectively; blue

circles, chromosomes. C: In S. cerevisiae at S phase, the half bridge (HB) elongates and satellite material is deposited into a nascent SPB next to

the mother SPB. At S/G2 phase, the daughter SPB reaches maturation. At metaphase, the SPBs move to opposite sides of the NE, becoming the

spindle poles. In red, SPB and HB; green lines, MTs; light green full lines, NE; Blue circles, chromosomes.D: InS. pombe, a bridge-like structure is

formed in G2 phase and a new SPB is formed at the distal end of the bridge at preprophase. The NE invaginates and forms a fenestra into which

the two SPBs settle. At metaphase, the SPBs move to opposite sides of the NE, becoming the spindle poles. In red, SPB and bridge-like structure;

green lines, MTs; light green full lines and dotted lines, NE and fenestra, respectively; blue circles reorganisation, chromosomes. E: In

D. discoideum at prophase, the layered structures increase in size along their axes. At prometaphase, the corona and cytoplasmic MTs disperse,

then the middle layer of the three-layered core structure disappears and the two outer layers peel apart. The two outer layers become the spindle

poles. In red, MTOC; green lines, MTs; light green full lines, NE; blue circles, chromosomes; black dotted lines, corona. For each organism only

the most important steps during cell cycle are shown.


BioEssays 31:874–884, � 2009 Wiley Periodicals, Inc. 877

Figure 3. Schematic representation of spindle formation, elongation and cytokinesis in diatoms compared to different model organisms.A: The

appearance of the PC (red rectangle) takes place at the beginning of preprophase close to the MC (red circle) outside the nucleus. The MC

disappears as the spindle elongates. By the end of prophase the spindle enters the nucleus but no complete NEBD occurs. At metaphase

chromosomes assemble around the spindle in a characteristic donut shape. At the end of anaphase B the cleavage furrow splits the fully

elongated spindle in two. B: In animals, centrioles (red rectangle) divide and the mitotic spindle elongates. At prometaphase NEBD occurs. At

metaphase chromosomes align between the poles of the spindle, then segregate. At telophase the cleavage furrow splits the spindle and two

daughter nuclei are reformed.C, D: In fission (C) and budding yeast (D), the SPB (red rectangle) divides and the spindle is generated between the

SPB pair sitting across the NE. No NEBD occurs. Cytokinesis takes place by fission in (C) and by budding in (D). E: In plants, MTs are nucleated

from numerous foci on the cortex in interphase. At preprophase foci condense at the centre to form the preprophase band. NEBD occurs.

Chromosomes align at metaphase. At telophase an outgrowing cell plate develops between the segregated chromosomes and splits the spindle.

In red, MTOCs; green lines, MTs; blue circles, chromosomes. Light green full, interrupted and dotted lines: NE, partially broken down NE and

broken down NE, respectively. Thin black lines, valves; thick black lines, cell wall, in grey, plasma membrane; black ring, contractile ring; violet

hemi-circles, polar cap.


diatoms. We have recently identified several unique cyclin/

CDKs and other cell cycle regulators in P. tricornutum, and

diatoms contain a large number of diatom-specific cyclins.(5)

Hinting at functions of some molecularcomponents of the diatom MTOC

Although high-quality EM has been carried out on MC and PC

in diatoms, almost nothing is known about their composition at

the molecular level. These two structures have the same

function, namely MT nucleation, but they do not look the same

and they act at different times of the cell cycle, suggesting that

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they could have different compositions and may be regulated

differently. From in silico analysis of the genomes of the

centric T. pseudonana(3) and the pennate P. tricornutum(5)

(http://genome.jgi-psf.org/Thaps3/Thaps3.home.html; http://

genome.jgi-psf.org/Phatr2/Phatr2.home.html), putative com-

ponents involved in the cell cycle regulation of MTOC and

the mitotic spindle can be identified (Supporting Information

Table S2).

Both the centrosome and SPB contain a g-tubulin (g-tub)

complex that serves as a template for polymerisation of

MTs(33) and establishes their polarity, with stable minus ends

facing the centrosome/SPB and plus ends facing outward.(34)

Although the g-tub complex and the nucleation mechanism


Figure 4. Four-dimensional confocal imaging of EB1-YFP-expres-

sing cells of P. tricornutum. The cell has been co-transformed with

EB1-YFP and CFP-H4 (a nuclear marker). The nucleus is labelled in

blue (CFP-H4 marker), the chloroplast in red (autofluorescence of

the pigments), and EB1-YFP in green. EB1-YFP can be seen to

localise to one central spot close to the nucleus that likely corresponds

to the MC, and from which MTs radiate to the cortex and around the

chloroplast of the cell. Scale bar: 2mm.


seem to be conserved, the mechanism for the organisation

of this complex varies in eukaryotes, with higher complexity

in mammals.(35) The three major conserved components

of g-tub complexes are g-tub, g-tub complex protein

GCP2 (Spc97p in S. cerevisiae) and GCP3 (Spc98p in S.

cerevisiae).(32,34,36) In the genomes of the two diatoms, genes

encoding g-tub, a GCP2-like and a GCP3-like protein have

been identified (Supporting Information Table S2).

Centrins are highly conserved components of the MTOC

playing an essential role in the duplication of the centrosome

and the SPB.(37) Several genes encoding centrin homologues

are present in the genomes of the two diatoms. Unlike other

algae studied so far, the genomes of T. pseudonana and

P. tricornutum were found to contain three genes encoding

centrins, one closer to animal orthologues and the other

closer to algal and plant sequences (Supporting Information

Table S2).

In both diatom genomes, we have also identified one end-

binding protein 1 (EB1) homologue (Supporting Information

Table S2). In mammalian cells EB1 is required for anchoring

cytoplasmic MTends to the mother centriole.(38) Its function at

MT plus ends is much better known, providing a physical link

between growing ends and the cell cortex and also with

kinetochores, through its interaction with adenomatous poly-

posis coli. Indeed, loss of this interaction leads to chromosome

instability in colorectal cancers.(39) In fission yeast, EB1 is a

good marker of early mitosis, and it is known to play a role in

spindle checkpoint control and is crucial for correct bipolar

kinetochore attachment.(40) InD. discoideum EB1 is an integral

component of the MTOC, part of the corona, and it may assist

in the initiation of MT outgrowth, but it is not required for MT

elongation.(32) In plant cells EB1 may also be involved in

anchoring MTs to their nucleation sites; in Nicotiana tabacum

BY-2 cells, EB1 appears to accumulate around the NE, in the

polar cap that survives NE breakdown (NEBD).(41–43)

To conclude, in diatoms three putative g-tub ring

complex proteins (g-tub, GCP2, GCP3), centrin-like proteins

and a putative EB1 represent conserved proteins found in

centrosomal and acentrosomal organisms. They could there-

fore be used as markers of MC and PC during the cell cycle,

like in other model organisms. EB1 is a good marker for MT

dynamics and nucleation in plants and animals and we have

recently developed 5D time-lapse confocal imaging of

P. tricornutum cells and have shown that the protein localises

at the MC and along the MTs in EB1-yellow fluorescent protein

(YFP)-expressing transgenic cells (Fig. 4). This and additional

markers could be used to better understand how the MC and

the PC cooperate during the cell cycle in diatoms.

The MTOC is a key component of cell cycle regulation.

Progression through the cell cycle is highly regulated by a

cascade of phosphorylation and dephosphorylation events,

specific to each phase of the cell cycle.(44,45) Some orthologues

of proteins involved in these processes have been identified in


the genomes of the two diatoms and may be of use to explore

cell cycle regulation in diatoms (Supporting Information Table

S2). For example, an Aurora-A homologue could be an

interesting candidate. Aurora-A kinase is essential for entry

into mitosis, and is required for cyclin B1/CDK1 activation at

the centrosome. It co-localises to centrosomes during

interphase and at both spindle poles and spindle MTs during

early mitosis up to anaphase.(46) It is required for centrosomal

spindle assembly, as well as kinetochore function.(47)

An unusual mitotic spindle andchromosome organisation in diatoms

The different phases of mitosis in diatoms have been well

documented by EM analysis (see Introduction, Fig. 2,

Supporting Information Table S1). One important feature of

diatom mitosis is that it is initiated outside the nucleus (closed

mitosis) when PCs are produced and the NE stays intact until

the elongating spindle enters the nucleus at prometa-

phase(10,31,48–50) (Fig. 3). By contrast, metazoans are char-

acterised by an open mitosis.(51) In yeasts, slime mould and

some protists, the NE tends to stay intact and MTs penetrate it

to have access to the chromosomes.(52–54) In higher plants,

prometaphase is characterised by NEBD.(55)

The central spindle in diatoms is composed of two

interdigitating portions made of antiparallel MTs (from a few

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units to hundreds), like in other organisms.(55,56) It is clearly

visible in EM, with a very well structured overlapping region

showing a unique paracrystalline arrangement, yielding the

conclusion that some bridge proteins might maintain the

structure(57–59) (Fig. 3A), as proposed for plant mitotic

spindles.(55) A set of motor proteins could act at the

overlapping region to pull apart the interdigitating MTs

during mitosis in diatoms (see below and(59)). At metaphase,

the chromosomes attached to the spindle have a unique

‘ring arrangement around the spindle equator’ that gives

the chromatin a characteristic ‘donut shape’(30,31) (Fig. 3A).

At this stage a dense matrix is visible in the spindles

of different diatoms (Fig. 3, Supporting Information

Table S1(30,48,57,60,61)). The so-called ‘collar material’ lies at

the chromosome-MT attachment site (see below).

Spindle elongation has been thoroughly studied in the

centric diatom S. turris,(11–13,50,59,62–64) and in the pennates

C. fusiformis(14–16,65,66) and Hantzschia amphioxys(67) using

different approaches. The spindle has been proposed to

elongate at anaphase B by different processes.(68) The

following evidence indicates that the main actor in spindle

elongation is the overlapping region: (i) biotinylated tubulin

incorporation studies showed that MT polymerisation occurs

mainly at the level of the overlapping region(11,64); (ii) a

reduction of the overlap was observed during mitosis (see

Fig. 3 in(59)); thus the overlapping region seems not to be a

fixed anchoring point for the two half-spindles but to play a

dynamic role as well as a structural one; (iii) in vitro studies on

isolated spindles showed that spindles elongate anyway even

though no cytoplasm surrounds the poles, and even after

chromatin digestion.(62,69) This means that the forces for

elongation do not reside in the PC or do not act on them but

are intrinsic to the spindle itself.

While the overlapping region is certainly a site for MT

polymerisation, it has been shown in vitro that isolated spindles

elongate even in the absence of tubulin (S. turris(11,50)) or in the

presence of MT polymerisation drugs,(62,70) and that anaphase

B movement is not affected in the presence of mitotic

inhibitors.(71) MT polymerisation can therefore be a permissive

feature but not a substantial one for spindle elongation itself.

More important is likely to be MT sliding as demonstrated

in vitro by Cande and coworkers.(12,13,68)

By coupling TEM micrographs with micro-cinematography,

it was shown that kinetochores in the pennate diatoms

Hantzschia and Nitzschia have a tri-plate structure, and

evidence based on EM observations was given to show that

they do not nucleate any MTs,(49) in contrast to what some

claimed at that time.(72) In diatoms the different MT types are

spatially separated, and the MTs involved in structural

maintenance of the central spindle do not attach to chromo-

somes and vice versa. After spindle entrance into the nucleus,

the chromosomes start oscillating back and forth to one or the

other pole possibly in order to orient their kinetochores to each

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pole(49) (chromosome congression(73)); then they segregate. A

unique feature observed in Surirella(30) and Pinnularia(61) is an

ill-defined matrix, called the collar, permeating the central

spindle mid-way between the poles and chromosomes. In

these species no conventional kinetochore fibres were

observed (Supporting Information Table S1).

To conclude, diatoms display several advantages for

studying the mitotic spindle: (i) some species are very large

and mitosis is rapid, enabling mitotic activity to be seen in living

cells with great clarity; (ii) they create a conspicuous central

spindle to which chromosomes attach; thus, unlike conven-

tional spindles, it is conceptually easy to see what structure

chromosomes attach to and move over during mitosis, and (iii)

their spindles are well organised, allowing their structure to be

analysed and correlated with their behaviour.

Glimpsing molecular features andregulation of diatom spindle andkinetochores

Several homologues of animal mitotic checkpoint and spindle-

associated regulatory proteins can be identified in the two

diatom genomes (Supporting Information Table S2). One

homologue of the kinetochore protein ZW10, which binds

the centromere and the kinetochore and is required for

chromosome segregation and for correct metaphase check-

point control, is present in P. tricornutum and T. pseudonana.

We have also identified several chromosome-associated

kinesins (chromokinesin) and several extra-spindle pole-like

proteins important for correct chromosome segregation

(Supporting Information Table S2). One is homologous to

the MAP65_ASE1 protein family, a putative MT-associated

protein that is essential for anaphase spindle elongation.

In animal cells there is a direct link between cell cycle

regulatory proteins and the assembly of the mitotic spindle,

which requires a protein complex that cross-links MTs

of opposite polarity.(74) A KRP called diatom spindle

kinesin 1(15,16) has been identified and characterised in

C. fusiformis as a motor protein involved in MT sliding.

Orthologues are present in both P. tricornutum and

T. pseudonana and could be good candidates to study the

spindle elongation process. However, in most of the previous

reports the presence of dynein was claimed instead of

KRP,(62) based on phosphorylation studies of vanadate-

treated in vitro isolated spindles.(12) Several dyneins are

encoded in the genomes of both diatoms.(3,4)

Kinetochores are multi-protein complexes that assemble

on centromeres and have three main functions: binding of

MTs, coordinating chromosome movements, and activating

spindle checkpoints. Centromeric regions vary in different

eukaryotes, from the 125-bp point centromeres in the budding

yeast(75) and the approximately 5-Mb human ones(76) to the



holocentromeres in the nematode Caenorhabditis ele-

gans.(77) Albeit different in size and arrangement, centromeric

organisation is commonly associated with rapidly evolving

satellite sequences.(78) But the spontaneous birth of new

centromeres (neocentromeres) supports the idea that

centromeres are epigenetically specified and maintained,

generating a modification in chromatin structure(79) that is

inherited via meiotic drive.(80)

Despite low conservation of centromeric regions, centro-

mere or kinetochore proteins are highly conserved (at least

between metazoans and yeasts(81)). Centromeres likely carry

out the same function throughout eukaryotes, as do

kinetochores.(82) Among kinetochore proteins there is the

centromere-specific histone H3 variant, further referred to as

CENH3, according to Dawe and Henikoff.(83) CENH3

produces modified nucleosomes that line up on the chromo-

somes facing the pole.(84) This is where such nucleosomes

interact with the foundation kinetochore proteins(78) that in

turn recruit proteins involved in MTattachment and checkpoint

controls.(78) Dynamic binding of the MT to the kinetochores is

achieved through the motor proteins dynein and dynactin as

well as the KRP MCAK and CENP-E in vertebrates(85) and

several protein complexes such as the NDC complex (Ndc80/

Hec1-Nuf2-Spc24/25(86–88)), almost all of which are present in

diatoms (Supporting Information Table S2).

Kinetochores also play a substantial role in regulating the

progression from metaphase to anaphase. If the chromo-

somes are not properly aligned before segregation, aneuploid

daughter cells could be produced.(73) The spindle checkpoint

function is accomplished by a complicated orchestration of

different proteins(89) that sense free kinetochores and

transduce them into signals that inhibit the anaphase-

promoting complex/cyclosome.(89) A centromere-specific

histone H3 variant is present in P. tricornutum (Supporting

Information Table S2) but not in T. pseudonana, possibly

corroborating the hypothesis of a low level of similarity

of centromeric proteins even among diatoms (as hypothe-

sised for other groups(90)), but contrasting with what is

found in fungi, where all CENH3 proteins are orthologous.(91)

Notwithstanding the centrality of kinetochore function and

the molecular aspects underlying centromere organisation,

kinetochore construction, kinetochore-MT attachment and

spindle checkpoint activation are unclear in diatoms and to

our knowledge no major advances have been made since the

1980s.

Cytokinesis: a combination of plantand animal features

Diatom cytokinesis is a mixture of animal (cleavage furrow)

and plant (synthesis of new cell wall) features (Fig. 3). As has

been elegantly shown by Pickett-Heaps and Pickett-Heaps by


micro-cinematography (‘Diatoms: Life in glass houses’; http://

www.cytographics.com/pg-dia.htm), ‘cytokinesis occurs by

furrowing’(92) and, as in animal cells,(43) the two newly formed

daughter cells are split. ‘Cells undergoing cytokinesis . . . show

an [actin] band encircling the cell at the cleavage furrow’.(93)

This finding supports the hypothesis that an actin contractile

ring-like structure is involved in diatom cytokinesis like in

animals and yeasts.(94) An actin band is also involved in

diatom valve morphogenesis(93,95) surrounding the growing

edges of the silica deposition vesicle, a specialised organelle.

The silica deposition vesicle is generally positioned in the

middle of the furrow and grows outwards, and is often

adjacent to the newly ‘reforming’ MC (see Supporting

Information Table S1) and the Golgi apparatus.(93,96)

Recent studies of conserved protein families have

revealed a common basis for the mechanism of cytokinesis

in plants and animals.(43) A structure deriving from the spindle

midzone sits in the middle of the contractile ring in animals

(midbody) promoting the trafficking of vesicles towards the

ingressing furrow. In plants it is the phragmoplast, an MT/actin

structure that has the function of transporting Golgi-derived

vesicles to the site of cell plate assembly.(97) By contrast,

S. pombe divides by septation. At the end of mitosis, an

equatorial MTOC (distinct to SPB), called the EMTOC, forms

mid-way through anaphase, recruiting g-tub to the equatorial

F-actin ring before it constricts to cleave the cell into two.(98)

It seems that in diatoms actin plays a double role in cell

division: first during furrowing (as in animals), and later

on during cell wall deposition (as in plants), again highlighting

the chimeric nature of diatom regulatory processes and

cytological features.(3,5)

In animal cells the position of the mitotic spindle determines

the position of the furrow at anaphase,(99) while division zone

determination happens far before anaphase in plants. In plants

the preprophase band, a transitory MT structure, marks the

future localisation of the cell plate.(97) Among the different

models for cytokinesis, the budding and fission yeasts

represent rather unique examples. S. cerevisiae divides by

budding; i.e. a new cell is produced by the constriction of a

contractile ring. In fission yeast the contemporary deposition of

cell wall material and the action of a contractile ring-like

structure separate the two daughter cells by fission. Yeasts

also diverge from other organisms in that determination of the

division plane occurs far before mitosis, at the G1/S boundary

in S. cerevisiae and in G2 in S. pombe.(99) In the centric diatom

S. turris, the location of the plane of division is cortically

determined before the mitotic spindle is formed and it is totally

independent of the position of the nucleus. This evidence has

been proven by short-term spindle displacement experi-

ments.(92) Although cytokinesis in brown algae has been

relatively well studied (centripetal with the development of a

plasma membrane furrow),(26) we have not compared it with

diatoms because, in spite of being phylogenetically related to

881


them, they show divergent features such as a centriolar mitotic

apparatus similar to animals,(26) have a different cell wall, and

are multicellular.

Conclusions

At the end of the 19th century, diatoms represented

one of the best models for scientists interested in

cell division. Lauterborn,(9) and subsequently Pickett-

Heaps,(10,20,28,30,31,48,49,57,58,60,61,71,93,95,96,100–104)

Cande(11–16,50,55,59,62–70,92,105) and collaborators have been

the pioneers of cell division studies in diatoms. Their beautiful

studies represent high-quality optical and EM descriptions of

nuclear division, spindle development and chromosome

segregation. They revealed intriguing features unique to

diatoms, such as (i) two independent organiser centres, the

MC and the PC, each with a role during interphase and

mitosis, respectively; (ii) a special ring distribution of the

chromosomes around the mitotic spindle at metaphase and a

paracrystalline organisation of the mitotic MT arrays; (iii) the

apparent absence of conventional kinetochores and centro-

somes, and (iv) cytokinesis with characteristics specific for

animals (cleavage furrow), plants (cell wall neosynthesis) and

diatoms (silica valve neosynthesis).

These observations invoke many exciting questions that

will need to be clarified by molecular approaches, such as the

precise and specific roles of MC and PC, their specific

structures and interconversion during the cell cycle, and the

mode of chromosome attachment and segregation on the

mitotic spindle. Although it is disappointing that these

pioneering works have been ‘frozen’ for the past 20 years

because of the lack of molecular technologies, molecular

tools and genomic data are now in place for cell division in

diatoms to be re-examined. In addition, diatoms offer several

advantages: (i) they are single cells easily observed in the

microscope and with easily identifiable mitotic structures; (ii)

the cells are easy to grow in the laboratory, and some species

are very large and mitosis is rapid; (iii) molecular tools for

reverse genetics have been developed; (iv) two completed

genome sequences are available, one from a pennate diatom

and one from a centric diatom, and (v) time-lapse imaging of

single cells expressing fluorescent fusion proteins has been

developed in P. tricornutum.

Several authors have recently reviewed and underlined the

diversity of structures involved in MT and mitotic spindle

organisation in eukaryotes, focusing on the animal and green

lineages.(43) Because of the chimeric and specific features of

cell division in diatoms, it will be interesting to add them to

such comparative analyses. Information about the origin and

evolution of the cellular structures involved in cell division in

eukaryotes may also aid our understanding of proliferative

diseases such as cancer.

882

Moreover some diatom species (mainly belonging to the

pennate genus Pseudo-nitzschia) can produce the neurotoxin

domoic acid(106) that accumulates in shellfish and can pose a

risk for human health.(107) These and other algal bloom

phenomena are examples of diatom cell proliferation in

response to specific environmental cues, and so it is of

interest to understand how external stimuli can provoke such

a dramatic activation of cell division. It is all the more urgent to

address such questions given current interest in large-scale

iron fertilisation of the southern oceans for carbon seques-

tration.(108) In these scenarios, iron enrichment is intended to

provoke massive diatom blooms which will remove CO2 from

the atmosphere through photosynthesis and export it to the

ocean interior.

Finally, diatoms are receiving attention as a source of algal

biofuels because of their high lipid content.(109) Such

approaches will require improvements of cell growth and

division in commercial settings and so an improved under-

standing of cell division will facilitate the development of

targeted strategies to optimise large-scale culture. To

summarise, we believe the new tools now available to explore

diatom biology can lead to a renaissance of studies of cell

division, thus vindicating the previous studies from the last two

centuries.

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Mitosis in diatoms: rediscovering an old model for cell division

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