Molecular dissection of plant cytokinesis and phragmoplast structure: a survey of GFP-tagged proteins

Molecular dissection of plant cytokinesis and phragmoplaststructure: a survey of GFP-tagged proteins

Daniel Van Damme, Francois-Yves Bouget†, Kris Van Poucke, Dirk Inze* and Danny Geelen

Department of Plant Systems Biology, Flanders Interuniversity Institute for Biotechnology, Ghent University, Technologiepark

927, B-9052 Gent, Belgium

Received 14 July 2003; accepted 3 August 2004.*For correspondence (fax þ32 9 3313809; e-mail [email protected]).†Present address: Laboratoire Arago, UMR7628 CNRS, Universite Pierre et Marie Curie, BP 44, F-66651 Banyuls sur Mer cedex, France.

Summary

To identify molecular players implicated in cytokinesis and division plane determination, the Arabidopsis

thaliana genome was explored for potential cytokinesis genes. More than 100 open reading frames were

selected based on similarity to yeast and animal cytokinesis genes, cytoskeleton and polarity genes, and

Nicotiana tabacum genes showing cell cycle-controlled expression. The subcellular localization of these

proteins was determined by means of GFP tagging in tobacco Bright Yellow-2 cells and Arabidopsis plants.

Detailed confocal microscopy identified 15 proteins targeted to distinct regions of the phragmoplast and the

cell plate. EB1- and MAP65-like proteins were associated with the plus-end, the minus-end, or along the entire

length of microtubules. The actin-binding protein myosin, the kinase Aurora, and a novel cell cycle protein

designated T22, accumulated preferentially at the midline. EB1 and Aurora, in addition to other regulatory

proteins (homologs of Mob1, Sid1, and Sid2), were targeted to the nucleus, suggesting that this organelle

operates as a coordinating hub for cytokinesis.

Keywords: actin, cell division, cytokinesis, microtubule, phragmoplast.

Introduction

Cytokinesis is more complex in plants than in animals due to

the presence of a rigid external wall. Testimony to the diffi-

culties the cell wall imposes on the division process are the

preprophase band (PPB) and the phragmoplast, two cyto-

skeletal structures that are necessary to assure adequate

positioning and assembly of a new cell wall between the

separating sister nuclei (Verma, 2001). The PPB is a ring of

actin filaments and microtubules (MTs) arranged in bundles

at the cell periphery that surround the nucleus temporarily

prior to the onset of mitosis (Mineyuki, 1999). The role of the

PPB in cell division is unclear but its occurrence seems to

correlate with a landmark, presumably inserted into the

plasma membrane that dictates the place where the future

cell wall will connect with the mother wall. On the contrary,

the phragmoplast guides Golgi-derived vesicles containing

cell wall synthesis enzyme complexes to the cell center to

construct a callose disk that gradually expands toward the

mother wall. Currently, our knowledge on the identity of

the molecular players that are involved in the functioning

of the PPB and the phragmoplast is far from complete.

Genetic screens of Arabidopsis thaliana plants have

identified several mutants with cytokinesis defects and, in

some cases, the affected genes have been isolated (Assaad,

2001; Smith, 2001). Interestingly, the genes identified in this

way were plant specific, such as tangled (Smith et al., 1996),

or were found to encode proteins with defined activities

related to vesicular trafficking and fusion (Assaad, 2001;

Bednarek and Falbel, 2002). Kinesins have been proposed to

be responsible for the transport of Golgi-derived vesicles to

the forming cell plate in addition to a role in maintaining the

integrity and organization of phragmoplast MTs (Liu and

Lee, 2001). Expansion of the cell plate requires the activation

of a MAPK signaling pathway mediated by the formation

and targeting of a MAPKKK–kinesin complex to the

phragmoplast (Nishihama et al., 2002).

Despite the uniqueness of the PPB and the phragmoplast

to plant species (Sawitzky and Grolig, 1995), a certain

structural resemblance can be recognized to ring-shaped

cytoskeletal organizations that occur in mitotic yeast and

animal cells. The organization of the phragmoplast is

reminiscent of the mid body in cytokinetic animal cells.

Both are composed of opposing bundles of MTs that face

386 ª 2004 Blackwell Publishing Ltd

The Plant Journal (2004) 40, 386–398 doi: 10.1111/j.1365-313X.2004.02222.x

one another with their positive ends at the center (Field

et al., 1999). However, the so-called actomyosin ring in yeast

and animal cells assists the cytokinesis process by recruit-

ment of membrane components and by a myosin-driven

contraction of the actin filaments in the ring, leading to a

narrowing of the division plane, also referred to as constric-

tion (Bi, 2001). Genetic studies in yeast have identified

numerous components of the contractile ring that function

in ring assembly, positioning, and contraction (Bi, 2001).

Because many of these proteins are evolutionarily con-

served in animals, common molecular mechanisms may

govern aspects of eukaryotic cell division (Feierbach and

Chang, 2001). However, it is not clear to what extent these

common factors occur in plants and, if so, what their

contribution to cytokinesis might be.

To investigate the alleged similarity of these cytoskeletal

structures and to build a comprehensive list of the compo-

nents involved, the Arabidopsis genome was searched for

cytoskeleton and cytokinesis-related genes. The selection of

genes and gene families analyzed was restricted to those for

which evidence of their implication in cytokinesis was found

in the literature. A recent review by Guertin et al. (2002)

gives an excellent overview of cytokinesis-related genes in

eukaryotes. Here, Arabidopsis actin-binding proteins (profi-

lins, formins, Rho-type GTPases, and myosins), MT-binding

proteins (MAP65, EB1, and CLIP170), regulatory proteins,

and putative cell cycle-controlled proteins were subcellularly

localized in interphase and dividing Bright Yellow-2 (BY-2)

cells of tobacco as well as in transformed Arabidopsis plants

by GFP tagging.

Results

Selection of putative cytokinesis-related Arabidopsis genes

Because cell division and cytokinesis are recurrent proces-

ses in all eukaryotes, accomplished by common underlying

mechanisms of signaling, membrane traffic, and cytoskele-

ton organization (Hales et al., 1999), we explored the

Arabidopsis genome sequence for homologs of known

cytokinesis genes by amino acid sequence BLAST searches.

Our survey pointed out that approximately one-third of the

53 genes and gene classes searched were conserved in the

Arabidopsis genome. The genes that were considered for

the analysis are listed in Tables S1–S4 (Supplementary

material available with this article online). The poor repre-

sentation of the known cytokinesis genes probably reflects

diversification in cytokinesis and division plane positioning

in plants. In particular, the lack of an actin or actomyosin ring

in plant cells is remarkable and fully in line with the absence

of genes encoding septins, type-II myosins, and IQGAP,

which are major components of the actin ring in yeast

and the cleavage furrow in animal cells (Guertin et al., 2002).

The emergence of an actin ring requires the inactivation

of the core cell cycle kinase that is mediated by anaphase-

promoting complex-controlled cyclin degradation

(Balasubramanian et al., 2000). Polo kinase coordinates the

mitotic and cytokinetic events by stimulating the proteolytic

activity of the anaphase-promoting complex and takes care

of proper positioning and assembly of the actomyosin ring

through phosphorylation of the Mid1 protein and associ-

ation with the septins cdc11 and cdc12 (Paoletti and Chang,

2000). Neither Polo kinases nor genes homologous to the

interacting Mid1 were found in the Arabidopsis genome,

suggesting that nuclear division and spatial control of

cytokinesis are not coupled in plants or rely on an alternative

regulatory mechanism.

The contractile ring structure is composed of actin

filaments and contains besides septins, IQGAP, and myosin,

also actin-associating proteins, including profilin, formin,

fimbrin, and the arp2/3 complex proteins. Although no

actomyosin ring is produced in plant cells, actin organization

is important for the completion of cytokinesis (Wick, 1991).

By using conserved sequences of the respective actomyosin

constituents, we identified several putative Arabidopsis

homologs (Table S1). Formin and profilin have been

assigned functions more related to the dynamics of actin

polymerization and are present in Arabidopsis as small

protein families consisting of 21 formins and four profilins

(Cvrckova, 2000; Deeks et al., 2002). Myosin-like sequences

were retrieved, but were neither of type II nor type IV

myosins, which are specific for the contractile actomyosin

ring. All 17 myosin-like Arabidopsis sequences put forward

so far belong to the type VIII and XI class, and perhaps a

third, ill-defined subfamily (Reddy and Day, 2001). We also

searched for GTPase-activating proteins, because small

GTPases of the Rho, Rac, and Cdc42 family and Rho-type

GTPase-activating proteins are required for cytokinesis in

animal cells (Table S2). The Rho-type Arabidopsis AtROP1

interacts with a callose synthase complex at the phragmo-

plast (Hong et al., 2001).

Because MTs are prominent components of the PPB and

the phragmoplast, we searched for MT-binding proteins.

Particularly, MT plus-end-binding proteins of the EB1 and

CLIP170 family are important for the establishment of

cellular polarity and the dynamics of MT structures. Three

EB1-like open reading frames (ORFs) were identified

(Table S1). We also looked for MAP65/Ase1/PRC1-like pro-

teins, a new class of MT-binding proteins of which Arabid-

opsis has nine members (Table S2; Hussey et al., 2002;

Schuyler et al., 2003). Two ORFs were found with similarity

to the spindle pole body protein Sad1þ from yeast. This

protein also associates with MTs and the nuclear membrane,

and may therefore have a task in coordinating the cytoske-

leton–membrane interphase (Hagan and Yanagida, 1995).

To test and evaluate GFP localization of Arabidopsis

proteins for which no function has been assigned based

on sequence information, we selected the homologs of

GFP survey of cell division genes 387

ª Blackwell Publishing Ltd, The Plant Journal, (2004), 40, 386–398

24 tobacco cDNA tags (indicated as T and a number;

Table S3) that have been shown to be cell cycle modulated

in synchronized BY-2 cells (Breyne et al., 2002). The corres-

ponding ORFs of seven S/G2-phase and 17 G2/M-phase

expression tags were selected for cloning and analysis

(Table S3).

In total, 103 Arabidopsis ORFs were selected for cloning.

The genes selected and yeast cytokinesis-related genes that

had no obvious homologs in Arabidopsis are listed in

Tables S1–S4. For 75 ORFs, we obtained GATEWAY-adapted

PCR fragments by using cDNA from an Arabidopsis cell

suspension as template. The PCR fragments were cloned in

frame with GFP placed at the C-terminal end, unless stated

otherwise. Expression of the fusion proteins was driven by

the 35S promoter. For some of the ORFs also N-terminal GFP

fusions were generated. Upon introduction of the constructs

into BY-2 cells, microscopic analysis identified 56 transgenic

lines with GFP fluorescence. The fluorescence patterns of

these lines, in addition to information on the different

constructs that were produced, can be found via links

accompanying Tables S1–S3 at our website (additional data

online: http://www.psb.ugent.be/papers/cellbiol).

GFP-fusion proteins localized to the nucleus, chromosomes,

and the nuclear envelope

The S and G2 phases are marked by changes in gene

expression and nuclear DNA structure reorganizations

(Heslop-Harrison, 2003). Chromosomes were labeled in

dividing Arabidopsis and BY-2 cells transformed with a

construct containing the S phase-specific T9 gene

(At5g11860) (Figure 1a,c,e). T9-GFP fluorescence was

concentrated in five to 10 nuclear dots in Arabidopsis

(Figure 1a,b) and many dots in BY-2 (Figure 1f). These nuc-

lear dots corresponded to regions of the nucleus that were

also heavily stained with 4,6-diamidino-2-phenylindole (data

not shown). According to the size of the nuclei and the

timing of appearance, the chromosomal arrangement in five

fluorescent dots seemed to occur in G1 cells and in 10 dots in

G2 cells (Figure 1a,b). During anaphase, the T9-GFP label

comigrated with the chromosomes to the poles (Figure 1c).

In the reassembled nuclei, again five dots were observed

(Figure 1d).

Several kinases (Aurora, Dyrk), phosphatases (PP2A), and

putative mitotic exit network (MEN)/septation initiation

network (SIN) components (AtSid1, AtSid2, and AtMob1)

were also targeted to the nucleus (additional data online). In

yeast, the spindle pole body operates as a signaling center

during cytokinesis (Simanis, 2003). MEN/SIN regulators,

such as Sid1 and Mob1, as well as Polo kinase temporarily

associate with the spindle pole body at some point in the cell

cycle. In analogy to this function, centrosomes have recently

been implicated in completing cytokinesis (Doxsey, 2001).

Because plant cells do not possess a spindle pole body or

centrosomes, the nuclear targeting of Sid1 and Mob1 can be

taken as an argument to put forward the nucleus as an

alternative center for the coordination of cytokinesis.

In this regard, the localization of GFP fusions of AtSad1a

(At5g04990) and AtSad1b (At3g10730), two Arabidopsis

homologs of the spindle pole body protein Sad1 (Hagan

and Yanagida, 1995), was informative. They brightly stained

the nuclear envelope in BY-2 and Arabidopsis cells

(Figure 2a,b). In dividing cells, AtSad1a-GFP strongly accu-

mulated in dots that were associated with the nuclear rim

or in the cytoplasmic space close to the plasma mem-

brane (Figure 2b,c). Upon nuclear envelope breakdown,

Figure 1. Targeting of T9-GFP to chromosomes and chromocenters.

(a) Arabidopsis root epidermal cells showing a metaphase cell with lined-up

chromosomes and nuclei containing five fluorescent nuclear dots.

(b) Arabidopsis root epidermal cell containing 10 fluorescent nuclear dots.

The nucleus is approximately twice the size of that of G1 nuclei.

(c) Anaphase cell in Arabidopsis root epidermal layer (arrow). Fluorescence is

concentrated at the chromosomes.

(d) Same cell as in (c) after completion of cytokinesis (arrows). T9-GFP

fluorescence is concentrated in five nuclear dots.

(e) BY-2 metaphase cell showing fluorescence of T9-GFP concentrated at the

chromosomes.

(f) Nuclear localization of T9-GFP in BY-2 nucleus and in nuclear foci.

Scale bars ¼ 5 lm (a,b), 10 lm (c,d,f), 20 lm (e).

388 Daniel Van Damme et al.


AtSad1a-GFP surrounded the spindle and the phragmoplast.

In addition, we reproducibly found that metaphase cells

contained one or a few, very bright fluorescent spots at one

end of the spindle and one or more spots at the other end,

attached to the plasma membrane (Figure 2c). Although

plant cells have no centrioles, the spots bear resemblance to

Sad1 bodies described in fission yeast mutants affected in

meiosis-specific spindle pole body integrity (Jin et al., 2002).

In plant cells, the nuclear surface is strewn with MT nuclea-

tion centers containing c-tubulin, which become polarly

organized when spindle formation begins (Binarova et al.,

2000; Schmit, 2002). These nucleation centers also contain

AtSpc98, a homolog of the yeast spindle pole body protein

Spc98 (Erhardt et al., 2002). In view of the role of the spindle

pole body and the centrosomes in establishing a bipolar

spindle and in coordinating events leading up to cytokinesis

(Doxsey, 2001), the nucleus and nuclear surface seemingly

play an important role in taking on these tasks in plant cells.

MT-binding proteins MAP65 and EB1

Arabidopsis contains nine MAP65-like genes (Hussey

et al., 2002) of which seven (AtMAP65-1, AtMAP65-2,

AtMAP65-3, AtMAP65-4, AtMAP65-5, AtMAP65-6, and

AtMAP65-8) were cloned and analyzed. Expression of

GFP-tagged proteins of AtMAP65-1, AtMAP65-3, AtMAP65-

5, and AtMAP65-8 resulted in fluorescent labeling of the

cortical array, an MT structure that is unique to plant cells

(Hardham and Gunning, 1978). Collapsed Z-stack images

of labeled cortical arrays of representative interphase BY-2

cells are shown in Figure 3(a–d). The MAP4 MT-binding

domain (MBD) and tubulin TUA6 were used as reference

for the fluorescent marking of MTs (Granger and Cyr,

2000; Hasezawa et al., 2000). Striking differences in labe-

ling patterns were observed. The labeling pattern in BY-2

cells containing the AtMAP65-5-GFP construct resembled

best the cortical MT array visualized by the MBD-GFP

control (Figure 3c). AtMAP65-1-GFP, on the contrary,

labeled much thicker bundles of cortical MTs and con-

centrated in dot-like structures that were attached to the

apparent end of the MTs (Figure 3a, arrows). These dots

were fixed in position over a period of 20 min and may

correspond to anchor points in the cell cortex, perhaps

connected to the plasma membrane (Hardham and

Gunning, 1978). AtMAP65-3-GFP fluorescence was very

pronounced at the centrally located endocytic MTs that

formed very thick bundles in comparison with the poorly

stained, much thinner cortical MTs. The endocytic MT

bundles wrapped around the nucleus and formed exten-

sions that were connected to the cortical MTs of the cell

poles (Figure 3b). Similar structures could also be ob-

served in transgenic BY-2 cells transformed with other

MAP65 constructs, the GFP-MBD, and TUA6-GFP controls.

However, in these cases, the cells were always committed

to enter mitosis. The constitutive and high expression of

AtMAP65-3-GFP may therefore have led to the persistence

or formation of these endocytic MTs. AtMAP65-3-GFP

caused extensive bundling of MTs at the center of the

cell, leaving cortical arrays within the same cell appar-

ently unaffected (Figure 3b). The endocytic MTs must

therefore have a different composition allowing for the

differential binding of AtMAP65-3-GFP. Another labeling

pattern was seen with the AtMAP65-8-GFP construct.

Whereas AtMAP65-3-GFP and AtMAP65-5-GFP labeled

the MTs homogenously, AtMAP65-8-GFP fluorescence

appeared as dotted lines (Figure 3d).

The punctate labeling of cortical MTs was also observed in

cells expressing AtEB1a and AtEB1b GFP-fusion proteins

Figure 2. Localization of AtSad1a to the nuclear envelope and Sad1-like

bodies.

(a) Confluent labeling of the nuclear membrane by overproduction of the

AtSad1a-GFP fusion product. Due to nuclear invaginations, knot-like struc-

tures penetrate the nucleus.

(b) Concentration of AtSad1a-GFP in dots that are in the cytoplasm and at the

nuclear rim. Cells are leaving the mitotic phase.

(c) BY-2 cell at metaphase. AtSad1a-GFP-labeled dots that are at the polar

ends of the spindle (arrow). The distribution is reminiscent of that of the

spindle pole bodies and associated Sad1 protein in dividing yeast cells.

Scale bars ¼ 10 lm (a,b), 20 lm (c).



(Figure 3f; Chan et al., 2003 [AtEB1a: At3g47690]; Mathur

et al., 2003 [AtEB1b: At5g62500]). AtEB1a and AtMAP65-8

concentrated at the spindle poles, suggesting that they are

associated with the minus-ends of MTs or the MT-organizing

centers (Figure 3e; Chan et al., 2003). The mouse adeno-

matous polyposis coli-binding protein EB1 localizes to

centrosomes where it could serve as an anchor point for

MT minus-ends (Askham et al., 2002).

However, the localization patterns of EB1 and the yeast

homolog BIM1 have also established them as MT plus-end-

binding proteins (Mimori-Kiyosue and Tsukita, 2003;

Mimori-Kiyosue et al., 2000; Schwartz et al., 1997). At the

plus-ends of MTs they recruit interacting factors involved in

an MT-capturing mechanism that influences MT dynamics

(Korinek et al., 2000; Lee et al., 2000). Fluorescence of the

dynamic plus-ends of MTs was visible as comet-like spots in

Arabidopsis epidermal cells that expressed AtEB1a-GFP or

AtEB1b-GFP (Figure 3f; additional data online; Chan et al.,

2003). Although the existence of a capturing mechanism in

plants has not been reported, it is likely that the plus-end-

bound AtEB1 proteins are involved in such a process

(Gundersen, 2002).

Remarkably, in contrast to AtEB1a and AtEB1b, AtEB1c did

not or very weakly label cortical MTs in interphase cells (data

not shown). Only with moderate to high laser excitation

intensities, AtEB1c was seen to bind MTs present at the cell

tip, and to some extent with the PPB MTs in prophase cells

(Figure 5a). The majority of the AtEB1c protein however was

concentrated in the nucleus (Figure 5a). During mitosis

AtEB1c was released from the nucleus upon nuclear envel-

ope breakdown and subsequently reentered the nucleus at

telophase (Figure 5a).

Actin-binding proteins

The actin-binding proteins that were analyzed did not

clearly label actin cables or the fine actin mesh. Differen-

tial interference contrast images of cells expressing actin-

binding GFP-fusion proteins often showed accumulation of

granular structures in the cytoplasm that are also observed

in damaged or stressed cells. Specific localization patterns

were obtained for GFP fusions of formin, profilin, and

myosin. GFP-fusion constructs of the type-I formins AtFH6

(At5g67470) resulted in fluorescent labeling of cross walls of

filamentous BY-2 cells (Figure 3g). In Arabidopsis roots and

Figure 3. Subcellular localization of cytoskeleton proteins in interphase cells.

(a–d) Projections of Z-stack confocal images, approximately two-third in the Z-

axis of transgenic BY-2 cells.

(a) Labeling of cortical microtubules (MTs) by AtMAP65-1-GFP and concen-

tration at MT ends, suggesting a preference for distal end labeling. Arrows

indicate dot-like structures at MT ends.

(b) Association of AtMAP65-3-GFP with thick bundles of endocytotic MTs that

emanate from the nucleus to connect with the cortical array at the cell poles.

The cortical MTs were much less stained and were visualized by increasing

the laser intensity of the confocal microscope. The cortical MTs appeared

thinner than those labeled with AtMAP65-1-GFP.

(c) Labeling of the cortical array by AtMAP65-5-GFP as a fine mesh of

transverse MTs. The labeling pattern was very similar to that of control cells

expressing MBD-GFP.

(d) Punctate accumulation of AtMAP65-8-GFP along MTs. The density of

visualized MTs was low compared with that of control lines, suggesting that

AtMAP65-8-GFP was associated with a subpopulation of MTs in the cortical

array.

(e) Bright fluorescence of AtMAP65-8-GPF at separate foci near the spindle

poles during mitosis (arrows). Some of the spindle MTs were also labeled.

(f) AtEB1b-GFP in Arabidopsis leaf epidermis. The plus-ends polymerize more

frequently than the minus-ends and become visible as comet-like structures.

Two stomata are shown in the left bottom corner.

(g) Labeling of the post-cytokinetic walls in BY-2 cell files by AtFH6-GFP.

(h) Optical section through an Arabidopsis root tip. AtFH6-GFP was primarily

targeted to the transverse walls.

Scale bars ¼ 20 lm (a–d, f and h), 10 lm (e).



stems, AtFH6-GFP fluorescence was present at the cell per-

iphery predominantly at the cross walls (Figure 3h). Type-I

formins are structurally different from their animal and

fungal counterparts in that they harbor an N-terminal signal

sequence followed by a transmembrane domain, which

could target these proteins to the secretory system

(Cvrckova, 2000; Deeks et al., 2002). AtFH6-GFP fluorescence

appeared exclusively at the periphery of the cell and may

therefore be associated with the plasma membrane.

Phragmoplast proteins

In total, 15 fusion proteins labeled the cytokinetic apparatus

(Figure 4; see additional data online). These proteins categ-

orized into functional groups as MT-binding, actin-binding,

signaling, and novel proteins. These proteins associated

with separate subregions of the phragmoplast and/or the

cell plate, demonstrating the existence of distinct structural

components of the cytokinetic apparatus. The most

important findings are summarized here.

The AtEB1a, AtEB1b, and AtEB1c proteins localized to the

phragmoplast MTs (Figure 4a; additional data online).

AtEB1a showed a preference for binding MTs near the

midline, suggesting they were concentrated at the plus-ends

of the antiparallelly arranged MTs, which can best be seen in a

collapsed image of a Z-stack series of the phragmoplast

(Figure 4a). During cytokinesis, all three AtEB1-GFP fusions

accumulated in highly fluorescent dot-like structures appear-

ing at the surface of the daughter nuclei and some dispersed

throughout the cytoplasm (Figures 4a and 5a; additional data

online). The dots that were in close contact with the nuclei

were usually larger than the cytoplasmic ones and they

mainly concentrated at the position where formerly the poles

of the spindle were located. After completion of cytokinesis,

the fluorescent dots disappeared. Subsequently, part of the

AtEB1c-GFP protein was redistributed to the extreme ends of

the BY-2 cell (Figure 5a). AtEB1c could therefore play a role in

the guidance of MTs toward the cell tips in agreement with

functioning in the capturing of MTs as proposed for the yeast

EB1 homolog BIM1 (Korinek et al., 2000; Lee et al., 2000).

Figure 4. Proteins targeted to the phragmoplast.

Confocal projections (a and b) and single optical sections (d–h) of BY-2 phragmoplasts. Arrowheads indicate the position of the equatorial plane.

(a) Labeling by AtEB1a-GFP of MT plus-ends that are assembled at the midline. In addition, brightly fluorescent dots occur in the cytoplasm and at the poles of the

daughter nuclei farthest from the equatorial plane.

(b) Strong AtMAP65-1-GFP fluorescence concentrated at the phragmoplast MTs and dots surrounding the nuclei. In contrast to AtEB1a (a), AtMAP65-1-GFP was

absent from MTs at the midline.

(c) Concentration of AtMAP65-3-GFP exclusively at the plus-ends of MTs in the midline.

(d) Accumulation of AtMAP65-5-GFP at the cell plate. An expanding phragmoplast is shown with the cell plate at the center and phragmoplast MTs at the border

(brace). Fluorescence is most pronounced in the region of the maturing cell plate as tubular structures, possibly plasmodesmata crossing the cell plate.

(e) End labeling of MT by AtMAP65-8-GFP that coresides with the MT-organizing centers located at the nuclear surface. The minus-ends of the phragmoplast MTs

were also labeled.

(f) Concentration of actin-binding protein myosin ATM1-GFP (AT3g19960) at the maturing cell plate. Fluorescence was evenly distributed, suggesting its association

with a membrane compartment.

(g) Concentration of T22-GFP at the cell plate and at the midline of the expanding phragmoplast ring.

(h) Concentration of At-Aurora 1-GFP at the young cell plate and at the position where formerly the spindle poles were.

Scale bars ¼ 20 lm.



AtMAP65-GFP proteins were also associated with

phragmoplast MTs but each had its own specific localization

pattern. AtMAP65-3 was strictly targeted to the midline and

did not bind MTs or MT ends that were located outside the

midline, in agreement with the immunolocalization data

recently reported by Muller et al. (2004; Figure 4c). On the

contrary, AtMAP65-1 and AtMAP65-8 were excluded from

the phragmoplast midline and labeled either along the MTs

(AtMAP65-1; Figure 4b) or at the outer extreme ends of the

phragmoplast MTs near the nuclear surface where the MT

minus-ends are located (AtMAP65-8; Figure 4e; Chan et al.,

2003). As AtMAP65-8-GFP also concentrated at foci at either

side of the spindle in metaphase cells (Figure 3e), this

protein seems to associate with MT minus-ends. In addition

to association with MTs, AtMAP65-1-GFP and AtMAP65-8

were concentrated in dots that surrounded the nuclei and in

the cytoplasm similar to those observed with the AtEB1

constructs (Figure 4b,e). AtMAP65-5-GFP showed a complex

localization pattern throughout mitosis and in particular

during development of the phragmoplast. AtMAP65-5-GFP

fluorescence was more intensely associated with the

phragmoplast than with the PPB and the spindle (Figure 5b).

It labeled the MTs from a young, emerging phragmoplast

but did no longer bind along the MTs when the disk-shaped

phragmoplast transformed into a ring-shaped, centrifugally

expanding structure (Figure 5b). The MTs of the ring-shaped

Figure 5. Time-lapse analysis of cytokinesis in BY-2.

Fluorescence of dividing BY-2 cells was imaged at time intervals indicated.

(a) Dynamics of AtEB1c-GFP localization. The highest concentration of the protein is present in the nucleus during interphase, but a small amount is observed at the

cell poles (full arrowheads). Upon envelope breakdown, AtEB1c was released and then immediately associated with the spindle microtubules (MTs) and

subsequently with the phragmoplast MTs. At the center of the developing cell plate, the MTs depolymerized. Some of the fluorescence remained at the nuclear

surface. When the nuclear membrane resealed, AtEB1c-GFP went partially back into the nucleus and partially moved to the junctions of the cell plate and mother wall

(open arrowheads) and to the cell tip (full arrowheads).

(b) Dynamics of AtMAP65-5-GFP localization. AtMAP65-5-GFP was excluded from the nuclear space until formation of the spindle. The phragmoplast MTs were

much more strongly labeled than the spindle. In the second phase of cell plate formation, which corresponds to a centrifugal expansion of the phragmoplast,

fluorescence persisted as focal points (plasmodesmata) at the matured cell plate. When cytokinesis was terminated some of the label was still present in the cross

wall (arrow). Parallel lines indicate the position of the preprophase band.




phragmoplast were not or poorly labeled, but instead

AtMAP65-5-GFP was concentrated in the cell plate and

associated with the surface of the separated nuclei (Fig-

ures 4d and 5b). During the phragmoplast expansion phase

the central MTs are depolymerized, callose is deposited, and

plasmodesmata are formed (Heinlein, 2002; Laporte et al.,

2003; Samuels et al., 1995). Because AtMAP65-5 remained

present in the cell plate after division was completed and

appeared to traverse the newly formed cross wall (Fig-

ures 4d and 5b), it is possible that AtMAP65-5 is an integral

part of plasmodesmata. Plasmodesmata are pierced with

MTs and indeed incorporate MT-binding proteins (Boyko

et al., 2002). For instance, GFP-tagged grapevine fanleaf viral

movement protein accumulates in the developing cell plate

of dividing BY-2 cells leading to the formation of fluorescent

tubular structures within the immature cross wall reminis-

cent of the structures labeled by AtMAP65-5-GFP (Laporte

et al., 2003).

In addition to microtubular structures, cell plate formation

also relies on actin and actin-dependent transport and on

fusion of Golgi-derived vesicles (Wick, 1991). We found that

the GFP fusions of the actin-binding protein myosin

(At3g19960) and an unknown protein T22 (At3g01780) were

primarily localized at the developing cell plate (Figure 4f,g).

T22 was most strongly associated with the leading edges

of the cell plate and followed the borders of the expand-

ing phragmoplast (Figure 4g). In contrast, myosin was

prominent throughout the cell plate and remained associ-

ated with the cross wall (Figure 4f). T22 was removed readily

from the post-cytokinetic wall, indicating that T22 and

myosin were differentially processed after completion of

cytokinesis (data not shown).

Finally, we identified three Aurora kinases in Arabidopsis.

The protein corresponding to At4g32830 (At-Aurora 1)

labeled spindle MTs (additional data online) and the cell

plate (Figure 4h). Higher eukaryotes have A-, B- and C-type

Aurora kinases of which the B-type is essential for cytokin-

esis (Carmena and Earnshaw, 2003). Overexpression of GFP-

tagged At-Aurora 1 led to the formation of binucleated cells

that were not observed in wild-type BY-2 or transgenic calli

transformed with either of the two other Aurora proteins

(Figure 6a). Because chromosome separation appeared

normal in the binucleated cells, At-Aurora 1-GFP must have

acted after chromosome segregation to inhibit cytokinesis in

a dominant negative manner. Binucleated cells were also

observed in transgenic cultures overproducing AtEB1c-GFP,

but not in cultures producing AtEB1a-GFP or AtEB1b-GFP

(Figure 6b).

Discussion

Because eukaryotic cells are highly compartmentalized, the

distribution and localization of a protein is intrinsically

bound to its function. With this principle in mind, screening

methods using random cDNA-GFP constructs to approach

plant gene function have been developed before (Cutler

et al., 2000; Escobar et al., 2003). The screening of such GFP-

fusion libraries requires a substantial input of effort with a

difficult-to-predict valuable outcome. To increase the

effectiveness of a GFP-based screening method we have

opted for a more selective cloning of genes related to cyto-

kinesis and cell division processes.

As the development of a cytokinesis program must have

had its origin early on in the evolution of the eukaryotic

lineage, we anticipate ample conservation of the implicated

molecular components that have previously been identified

through genetic analysis of yeast and animal cells (Feierbach

and Chang, 2001). However, merely a third of the genes

searched for have obvious homologs in the Arabidopsis

genome. Outstanding absentees are Polo kinase and several

of the structural components of the actomyosin ring, an

actin structure not formed by plants. The lack of a Polo

kinase is surprising, especially because one of its roles is to

trigger cytokinesis directly through the activation of the SIN

pathway in fission yeast (Song and Lee, 2001; Tanaka et al.,

2001). Most of the components that are part of the MEN/SIN

signaling machinery have homologs in Arabidopsis, sug-

gesting that at least this end of the regulatory pathway has

been conserved. Further support for the conservation of a

SIN-like pathway in plants follows from the complementa-

tion of a loss-of-function mutation in Saccharomyces pombe

Figure 6. Cytokinesis defects in transgenic BY-2 cells.

(a) Binucleated cell in At-Aurora 1-GFP line. Two nuclei lie adjacent to each

other without a separating wall. At-Aurora 1-GFP is present in the cytoplasm

and the nucleus and is concentrated at the nuclear rim.

(b) Binucleated cell in AtEB1c-GFP cell line. The brightness of the image was

increased to reveal fluorescence associated with endocytic microtubules

(MTs). A cell plate is missing in-between the two separated nuclei. Instead, a

few MTs connect the nuclei to each other. Fluorescent dots at the cell

periphery indicate the presence of a cortical array. Dense material at one side

of the cell is seen (arrow) that could be the remnants of an unsuccessful

attempt to make a cell wall.




of the MAP3K kinase SIN element cdc7p by a Brassica napus

homolog (Jouannic et al., 2001).

Our survey has generated a useful set of marker GFP-

fusions that are particularly relevant for the study of cell

division-related processes. For example, the T9 protein is a

unique marker that labels the nucleus and concentrates at

nuclear sites of condensed chromatin also referred to as

chromocenters (Fransz et al., 2002). These areas correspond

to the centromeric regions of Arabidopsis interphase chro-

mosomes (Fransz et al., 2002; Talbert et al., 2002). The

association of T9-GFP to the chromocenters can be exploited

to monitor chromatin organization according to the cell

cycle phase, either in single cell cultures or in cells imbedded

in the context of a whole tissue. The amino acid sequence of

T9 (At5g11860) indicates similarity with a nuclear LIM-

interacting protein (NLI interacting factor 1 or NIF1), a

human protein for which the function remains to be iden-

tified (Jurata et al., 1996).

The AtSad1a and AtSad1b GFP fusions are interesting

because of a potential connection between the nuclear

membrane and MT nucleation. Moderate overexpression of

Sad1 in yeast leads to association with the nuclear envelope

(Hagan and Yanagida, 1995). Fission yeast Sad1 protein is a

constitutive spindle pole body (SPB) component essential

for spindle formation and function. Plants do not possess

SPB or centrosome-like structures, but nevertheless harbor

genes homologous to the SPB elements Spc97, Spc98, and

Sad1 (Erhardt et al., 2002; this work). The AtSpc98 protein

colocalizes with c-tubulin at the cell cortex and the nuclear

periphery where it stimulates tubulin polymerization

(Erhardt et al., 2002). So-called Sad bodies that are not the

products of ordinal SPB duplication have recently been

found associated with the nucleus in zygotic yeast cells

(Goto et al., 2001). The occurrence of the Sad bodies, which

contained other SPB components, correlates with particular

centromere and telomere arrangements in meiotic prophase

nuclei, indicating that the functioning of these proteins is not

restricted to MT nucleation (Goto et al., 2001; Jin et al.,

2002). The AtSad-GFP concentrates in highly intense fluor-

escent dots located in the cytoplasm as well as dots at either

side of the spindle, sometimes associated with the cell

periphery. Because of the occurrence of fluorescent dots in

the cytoplasm, we suspect that the function of AtSad, as in

yeast, is implicated in a process other than that of MT

nucleation.

Despite poor conservation of cytokinesis genes, our

survey has identified 15 GFP-fusion proteins that are

targeted to the cytokinetic apparatus. The proteins

involved are members of the protein families EB1-like,

MAP65, myosin, formin, Aurora, Rho-like GTPases Rop,

and a novel protein of unknown function provisionally

nominated T22. The fluorescence pattern in BY-2 cells of

each of these proteins was highly distinctive and marked

different subregions of the cytokinetic apparatus. EB1 and

MAP65 proteins highlighted MTs with a preference for

either the plus-ends, the minus-ends, or along the length

of the polymers.

Recently, AtEB1a was reported to be a MT minus-end

binding protein and AtEB1a associates with slowly moving

dots dispersed in the cortical array that are believed to be

MT minus-ends based on their mobility properties (Chan

et al., 2003). As AtEB1a was also present at fast-growing

MT tips, it was proposed that AtEB1a has the capacity to

bind both MT plus- and minus-ends. Our data do not

confirm the association of AtEB1a-GFP with MT minus-

ends perhaps because a different suspension cell type was

studied (Arabidopsis versus BY-2). Alternatively, the

expression levels in stably transformed BY-2 cells may

have been more moderate compared with a transient

expression system used by Chan et al. (2003) and have

disallowed detection of minus-end association. For in-

stance, in the continuously propagated AtEB1a-GFP and

AtEB1b-GFP BY-2-expressing cell lines, we did not observe

label along MTs and the fluorescence at the MT ends were

1–2 lm in size rather than 2–5 lm as was reported (data

not shown; Chan et al., 2003; Mathur et al., 2003). In

agreement with the findings of Mathur et al. (2003) who

showed that AtEB1b, in addition to the MT plus-ends,

associates with the endoplasmic reticulum and the endo-

membrane system we found background fluorescence in

the cytoplasm that remained upon continuous propaga-

tion. Given that AtEB1a and AtEB1b were primarily plus-

end MT-binding proteins, the localization of AtEB1c-GFP in

the nucleus was unexpected. The AtEB1c-GFP MT-binding

capacity is revealed during mitosis when it is released

from the nucleus after envelope breakdown and it labels

the spindle and phragmoplast. Whether AtEB1c has a

preference for MT plus-ends was difficult to detect

because of the three-dimensional organization and density

of MTs in the spindle and the phragmoplast structures.

Because AtEB1c gets redistributed to the extreme tips of

the newly born cells when cytokinesis is completed, it is

possible that AtEB1c plays a role in polarized cell growth

similar to the MT plus-end-binding protein CLIP170 in

fission yeast (Feierbach et al., 2004).

AtMAP65-3-GFP was associated with phragmoplast MTs

near the midline and at the spindle midzone, suggesting that

AtMAP65-3 binds to overlapping MT plus-ends. In addition,

we show that AtMAP65-3-GFP labeled the cortical array

although the fluorescence was much weaker compared with

that of other MAP65-GFP fusions and the signal coming from

endocytic MTs. Immunolocalization of AtMAP65-3 with a

specific antibody does not confirm localization to the cortical

array or the spindle and showed an exclusive association

with the phragmoplast (Muller et al., 2004). As AtMAP65-3

transcription peaks at mitosis, protein synthesis is likely cell

cycle controlled and limited to the mitotic phase (Menges

et al., 2003). Continuous expression driven by the 35S



promoter may therefore lead to artificial accumulation of

AtMAP65-3-GFP at the cortical MTs. As AtMAP65-3-GFP was

not concentrated at the plus-ends of cortical MTs, suggesting

that the antiparallel arrangement of the MT plus-ends, as it

occurs in the phragmoplast midline and the spindle midzone,

is needed for strong MT binding. Biochemically purified

Nicotiana tabacum NtMAP65 protein cross-links in vitro

stabilized MTs (Chan et al., 1999). Antibody raised against

purified NtMAP65 protein binds to a small family of MAP65

proteins collectively called NtMAP65-1 that were localized to

the midline in cytokinetic BY-2 cells, in line with a role in

stabilization of the antiparallel phragmoplast MTs (Smerten-

ko et al., 2000). A similar stabilizing function was proposed

for Ase1 in yeast, a map65 homolog that is essential for the

establishment and maintenance of bipolar organization of

the spindle (Schuyler et al., 2003). In analogy to the function

of ASE1, NtMAP65-1 and AtMAP65-3 could act as anchors for

MT plus-ends at sites where they interdigitate, thereby

maintaining phragmoplast bipolarity. The absence of

AtMAP65-1-GFP, AtMAP65-5-GFP, and AtMAP65-8-GFP from

the midline indicates that cross-linking of MT plus-ends is not

a common feature of all members of the MAP65 family.

The localization of AtMAP65-8 was most remarkable, as it

concentrated at the outermost side of the phragmoplast as

well as at the spindle poles. Here, MT minus-ends congreg-

ate in multiple sites of MT-organizing centers from which

MTs emanate. The phragmoplast MTs are also polarly

organized and nucleate from sites near the surface of the

nucleus that faces the equatorial plane. The minus-ends of

MTs are anchored to the organizing centers so that new MTs

polymerizing at the plus-end originate from a fixed position.

In the cortical array, AtMAP65-8-GFP clearly stained along

MTs, but in contrast to GFP-MBD and TUA6-GFP controls,

the label was heterogeneously distributed. The MAP65-

8-GFP protein also concentrated at relatively immobile foci

in the cortical MT array, which may correlate with the

peripherically organizing centers in interphase cells previ-

ously described based on c-tubulin localization experiments

(Figure 3d) (Canaday et al., 2000). We therefore propose that

AtMAP65-8 is associated with MT-organizing centers both in

interphase and in dividing cells.

Although localization at the phragmoplast in itself is no

good proof, other evidence supports a role for the phragmo-

plast proteins in cytokinesis. Several of the phragmoplast-

targeted proteins were transcriptionally activated at mitosis.

For instance, the expression of Aurora and T22 peaks in

synchronized BY-2 cell cultures 7 h after aphidicoline

release, along with the expression of B-type cell cycle-

dependent kinase (cluster 1; Breyne et al., 2002). The RNA

levels of AtMAP65-3, AtEB1c, and Aurora significantly

increased during mitosis according to microarray analysis

of synchronized Arabidopsis cell cultures (Menges et al.,

2003). We noticed that in several transgenic BY-2 lines

aberrant cells carrying multiple nuclei occurred, indicative of

erroneous cytokinesis events. Cytokinesis defects were most

pronounced in cell lines expressing the cell cycle-controlled

genes AtEB1c, T22, and Aurora. The Aurora BY-2 lines that

most frequently produced binucleated cells gradually died

and could not be maintained for extended periods because

of accumulative defects in the division process probably due

to the constitutive expression of the fusion protein. Despite

constitutive expression driven by the 35S promoter, some of

the candidate cytokinesis proteins were primarily targeted to

the phragmoplast structure and not to other mitotic config-

urations. For example, AtMAP65-1-GFP and AtMAP65-3-GFP

were abundantly present in the phragmoplast and much less

in the spindle. The localization of AtMAP65-3 is in good

agreement with the cytokinetic phenotype of the Arabidop-

sis root morphogenic mutant pleiade that carries a null

mutation in the AtMAP65-3 gene (Muller et al., 2002).

Pleiade roots develop expanded irregularly shaped cells

containing multiple nuclei probably as a consequence of the

formation of distorted phragmoplasts with a widened mid-

line (Muller et al., 2004).

Fluorescence of candidate cytokinesis proteins was

mostly confined to particular subregions of the phragmo-

plast MTs or the cell plate, in line with the functional

divergence of the respective proteins. For example, the

novel protein T22 preferentially concentrated at the rim of

the expanding phragmoplast where Golgi-derived vesicles

arrive to build the new callose-containing cell wall (Samuels

et al., 1995). Myosin-GFP fluorescence, on the contrary, was

mostly pronounced at the developing cell plate after

removal of the phragmoplast MTs.

In conclusion, the GFP-fusion proteins identified in this

study represent valuable markers for future investigations

in plant cytokinesis and will be helpful in unraveling

structure/function relationship of the phragmoplast in live

plant cells.

Experimental procedures

Cell suspension cultures and transformation

Arabidopsis thaliana (L.) Heyhn. Landsberg erecta cells weregrown in 4.43 g Murashige and Skoog (MS) medium (Duchefa,Haarlem, The Netherlands), 30 g sucrose, 500 lg NAA and 50 lgkinetin per liter on a rotating platform (150 rpm) at 27�C in thedark. Nicotiana tabacum L. Bright Yellow-2 (BY-2) cells weregrown in 4.302 g MS, 0.2 g KH2PO4, 30 g sucrose, 0.02 mg2,4-dichlorophenoxyacetic acid (auxin), 0.05 mg thiamine, 5 mgmyo-inositol per liter, pH 5.8 under the same incubation condi-tions. Transformation procedure was as described (Geelen andInze, 2001). Multiple transgenic BY-2 calli were transferred to freshmedium and inspected for GFP fluorescence with an epifluores-cence microscope (Axioskop; Zeiss, Heidelberg, Germany). Ara-bidopsis was transformed by the floral dip method (Clough andBent, 1998). Primary seed transformants were selectively grownon 4.43 g l)1 MS, 6.5 g l)1 agar, pH 5.7 containing 75 lg ml)1

kanamycin.



Sequence identification and gene cloning

The Arabidopsis genome was interrogated by BLAST aminoacid sequence searches (http://www.ncbi.nlm.nih.gov/BLAST).Sequences were selected on an individual basis by consideringoverall similarity levels and the conservation of crucial amino acidpositions. The putative start and stop codons of the identified geneswere used for primer design (additional data online). For cloning,RNA was extracted from cells after 48, 60, and 72 h subculturing,following the method described by Leyman et al. (2000). Poly(A)þRNA was purified using oligo dT(25)-coated Dynabeads (Dynal,Oslo, Norway). ORFs were cloned by a one-step RT-PCR reaction(Titan one tube RT-PCR kit; Roche Diagnostics, Brussels, Belgium)with high-fidelity DNA polymerase and 0.1 lg purified poly(A)þ astemplate in 50 ll. Primers contained attB1 and attB2 extensions forGateway� (Invitrogen, Carlsbad, CA, USA) conversion and addi-tional modifications for replacement of the stop codon by a tyrosinecodon in frame with the enhanced fluorescent protein EGFP. PCRproducts were gel-purified (High pure PCR purification kit; RocheDiagnostics), and subcloned into pDONR207 via BP reaction cloning(Gateway�). Recombined plasmids (entry clones) from eight col-onies were analyzed by restriction digest (AvaI) and sequenceanalysis. The inserts of the entry clones were transferred to the plantGateway ‘destination vector’ pK7WGF2 (Karimi et al., 2002) togenerate EGFP C-terminal fusions downstream of the strong con-stitutive 35S promoter. Plasmids were checked by EcoRV restrictiondigest and transferred to Agrobacterium tumefaciens strainLBA4404.

Microscopy

Approximately 10 transgenic BY-2 calli of 1 lm were inspected forfluorescence under a coverslip with an Axioskop (Zeiss) fluores-cence microscope. In those cases where no EGFP fluorescencecould be detected, instability of the fusion protein, lack of expres-sion due to silencing, or counterselection were assumed (Joubeset al., 2003). GFP-positive calli and Arabidopsis seedlings wereanalyzed by confocal microscopy (Zeiss 100M, equipped withLSM510 software version 3.2). A 63X water corrected objective(numerical aperture of 1.2) was used to scan the samples. Theimages were captured with the LSM510 image acquisition software(Zeiss). Projections were obtained from approximately 30 serialoptical sections, 0.5 lm apart to cover two-thirds of the cell depth.Images were exported as TIFF files and processed with AdobePhotoshop (version 7). Three-dimensional and time-lapse confocalmicroscopy were carried out on BY-2 cells fixed with poly-L-lysine tothe bottom of an 8-well coverglass chamber (Lab-Tek, Naperville, IL,USA) containing 100 ll BY-2 culturing medium. Under these con-ditions, BY-2 cells carried on dividing and could be monitoredovernight.

Supplementary material

The following material is available from http://www.blackwellpublishing.com/products/journals/suppmat/TPJ/TPJ2222/TPJ2222sm.htm.Table S1 Arabidopsis genes similar to yeast genes that have beenimplicated in cytokinesisTable S2 Arabidopsis genes that encode potential cytoskeleton-associated proteinsTable S3 Arabidopsis genes that are the putative orthologs of cellcycle-modulated genes previously discovered by cDNA-AFLP ana-lysis in synchronized BY-2 cell cultures (Breyne et al., 2002)

Tables S1 and S3 include a list of gene name and proposed functionwith corresponding degree of similarity from the BLAST results, andTables S1, S2, and S3 provide the MIPS code, cDNA length, PCRamplification result, Gateway cloning, and epifluorescence detec-tion for the selected genes.Table S4 List of yeast genes for which no homologous sequencewas found in the Arabidopsis genome

Additional data online

A database of localization patterns linked with Tables S1, S2 and S3can be found at http://www.psb.ugent.be/papers/cellbiol.

Acknowledgements

We thank Martine De Cock for help with the manuscript. D.V.D. andD.G. are postdoctoral and predoctoral fellows of the Fund for Sci-entific Research-Flanders respectively. F.-Y.B. is supported by theCentre National de la Recherche Scientifique, France.

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Molecular dissection of plant cytokinesis and phragmoplast structure: a survey of GFP-tagged proteins

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