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The organization of the cytoskeleton during meiosis in eggplant {Solanum melongena (L.)): microtubules and F-actin are both necessary for coordinated meiotic division J. A. TRAAS 1 ' 2 , S. BURGAIN 1 and R. DUMAS DE VAULX 1 l INRA, BP94, 84140 Montfcivet, France* 2 I.V.T., Wageningen, The Netherlands •Address for reprints Summary Because two division planes form at right angles, male meiosis in higher plants provides striking examples of both division control and spatial pro- gramming. To investigate these processes we have stained microtubules and actin filaments during male mei- osis in the eggplant. Our results indicate the follow- ing. (1) That microtubules and their nucleation sites are involved in the establishment of polarity; this is supported by our observation that the drug CIPC affects spindle polarity. (2) That actin microfilaments are involved in spindle formation and integrity, but not in the establishment of polarity: cytochalasin B and D affect the organization of the spindle microtubules, but not their polarized distribution. (3) That microtubules radiating from the daughter nuclei at the cell poles during interkinesis probably establish the future division plane by concentrating actin in that plane (cf. the proposed role of asters in positioning the contractile ring in animal cells). (4) That this concentration of F-actin in the division plane may be involved in preparing the cytoplasm for cytokinesis and in memorizing the division plane (much as the preprophase band observed in polarized tissues does). (5) That phragmoplast formation is a two-step process. No phragmoplast forms after metaphase I, but a four-way phragmoplast forms after meta- phase II, indicating that mitosis and cytokinesis are not obligatorily coupled. These studies demonstrate that actin and micro- tubules are jointly involved in the spatial coordi- nation of the division process. Key words: cytoskeleton, eggplant, F-actin, microtubules, meiosis. Introduction Meiosis has been studied mainly from the 'chromosomal' point of view and much is known about the behaviour and configuration of the chromosomes (reviews: John & Lewis, 1965; Sybenga, 1975; Dickinson, 1988). Yet, the cytoplasmic mechanisms that control meiosis, determine chromosome pairing, define with great precision the division planes and ensure the distribution of cytoplasm between the daughter cells are of equal importance but remain poorly understood. Because of this, some atten- tion has been paid to the role of the cytoskeleton during male meiosis in higher plants and changes in microtubu- lar and, to a lesser extent, F-actin arrays have been described for a number of species (Van Lammeren et al. 1985; Sheldon & Dickinson, 1986; Hogan, 1987; Sheldon & Hawes, 1988). Such studies show that, as in normal Journal of Cell Science 92, 541-550 (1989) Printed in Great Britain © The Company of Biologists Limited 1989 somatic mitosis, microtubules function in nuclear div- ision, whereas in cytokinesis F-actin accompanies micro- tubules in forming the phragmoplast. However, a num- ber of important questions remain to be answered, especially concerning the establishment and maintenance of the well-defined division planes by which four haploid microspores are separated by two meiotic divisions. Recent findings have established that F-actin plays an important role in determining the division plane of somatic cells (Traas et al. 1987; Lloyd & Traas, 1988; Lloyd, 1988). Actin filaments had been thought to be absent during mitosis, but by avoiding aldehyde fixation (by detergent extraction or electroporation) it was dis- covered that a network of actin persists throughout mitosis and cytokinesis. The actin envelopes the nucleus and, by transvacuolar filaments, connects that organelle to the cortex. In somatic mitosis the division plane is 541

Cito Esqueleto Meiosis

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The organization of the cytoskeleton during meiosis in eggplant {Solanum

melongena (L.)): microtubules and F-actin are both necessary for

coordinated meiotic division

J. A. TRAAS1'2, S. BURGAIN1 and R. DUMAS DE VAULX1

lINRA, BP94, 84140 Montfcivet, France*2I.V.T., Wageningen, The Netherlands

•Address for reprints

Summary

Because two division planes form at right angles,male meiosis in higher plants provides strikingexamples of both division control and spatial pro-gramming.

To investigate these processes we have stainedmicrotubules and actin filaments during male mei-osis in the eggplant. Our results indicate the follow-ing.

(1) That microtubules and their nucleation sitesare involved in the establishment of polarity; this issupported by our observation that the drug CIPCaffects spindle polarity.

(2) That actin microfilaments are involved inspindle formation and integrity, but not in theestablishment of polarity: cytochalasin B and Daffect the organization of the spindle microtubules,but not their polarized distribution.

(3) That microtubules radiating from thedaughter nuclei at the cell poles during interkinesisprobably establish the future division plane by

concentrating actin in that plane (cf. the proposedrole of asters in positioning the contractile ring inanimal cells).

(4) That this concentration of F-actin in thedivision plane may be involved in preparing thecytoplasm for cytokinesis and in memorizing thedivision plane (much as the preprophase bandobserved in polarized tissues does).

(5) That phragmoplast formation is a two-stepprocess. No phragmoplast forms after metaphase I,but a four-way phragmoplast forms after meta-phase II, indicating that mitosis and cytokinesis arenot obligatorily coupled.

These studies demonstrate that actin and micro-tubules are jointly involved in the spatial coordi-nation of the division process.

Key words: cytoskeleton, eggplant, F-actin, microtubules,meiosis.

Introduction

Meiosis has been studied mainly from the 'chromosomal'point of view and much is known about the behaviour andconfiguration of the chromosomes (reviews: John &Lewis, 1965; Sybenga, 1975; Dickinson, 1988). Yet, thecytoplasmic mechanisms that control meiosis, determinechromosome pairing, define with great precision thedivision planes and ensure the distribution of cytoplasmbetween the daughter cells are of equal importance butremain poorly understood. Because of this, some atten-tion has been paid to the role of the cytoskeleton duringmale meiosis in higher plants and changes in microtubu-lar and, to a lesser extent, F-actin arrays have beendescribed for a number of species (Van Lammeren et al.1985; Sheldon & Dickinson, 1986; Hogan, 1987; Sheldon& Hawes, 1988). Such studies show that, as in normal

Journal of Cell Science 92, 541-550 (1989)Printed in Great Britain © The Company of Biologists Limited 1989

somatic mitosis, microtubules function in nuclear div-ision, whereas in cytokinesis F-actin accompanies micro-tubules in forming the phragmoplast. However, a num-ber of important questions remain to be answered,especially concerning the establishment and maintenanceof the well-defined division planes by which four haploidmicrospores are separated by two meiotic divisions.

Recent findings have established that F-actin plays animportant role in determining the division plane ofsomatic cells (Traas et al. 1987; Lloyd & Traas, 1988;Lloyd, 1988). Actin filaments had been thought to beabsent during mitosis, but by avoiding aldehyde fixation(by detergent extraction or electroporation) it was dis-covered that a network of actin persists throughoutmitosis and cytokinesis. The actin envelopes the nucleusand, by transvacuolar filaments, connects that organelleto the cortex. In somatic mitosis the division plane is

541

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predicted by the formation of a preprophase band (PPB)of microtubules, but it is now apparent that actinfilaments also form a cortical band, so that the PPB is nolonger considered to consist of microtubules exclusively.Even though the PPB microtubules disappear by meta-phase, the radial nucleus-associated actin strands remainin the division plane, providing a memory of the divisionsite and helping to guide the cytokinetic apparatus outalong the pre-determined path. The significance of theseobservations is that actin and microtubules combine toset up the division plane. In view of this, we have re-examined meiotic division, which differs from mitosis inseveral important aspects: there is no PPB, for example,and there is no known basis for the four-square position-ing of the haploid microspores produced by two success-ive meiotic divisions. However, in this paper we nowreport the presence of an equatorial system of F-actin thatpredicts the first division plane. As in mitosis, micro-tubule-actin interactions seem to be essential for thespatial control of male meiotic division.

Materials and methods

Plant materialTwo varieties of Solatium melongena (L.) were used for theexperiments: Ronde de Valence and Doutga. Plants were grownunder greenhouse conditions. Young buds were cut from theplants and the stage of meiocyte development was determined inone anther of each bud. For this purpose anthers were squashedin water and examined in an Olympus BH2 microscopeequipped with Nomarski optics. This gives a satisfactoryestimate of the stage of the four or five remaining anthers asmicrospore formation is highly synchronized within each bud.Only anthers containing meiocytes at a stage prior to divisionwere used. This stage is characterized by the formation of thethick callosic wall.

Anther culture and drug treatmentsIntact anthers were removed from the buds and put in 3-5 cmPetri dishes containing 2 ml solid 'T ' medium (pH 59) withouthormones (concentrations of macro- and micronutrients, vit-amins, sucrose and agar were as described by Chambonnet &Dumas de Vaulx, 1983). Under these conditions meiosisproceeds normally within 18 h of culture. The following drugswere used: chloroisopropylphenyl carbamate (CIPC) (Sigma),colchicine (Prolabo), taxol (a generous gift from NCI, Beth-esda), cytochalasin B and D (Sigma), and phalloidin (Sigma).The drugs were first solubilized in dimethyl sulphoxide(DMSO) and subsequently diluted in liquid T medium.Colchicine was also solubilized in water. The different concen-trations used for each drug are given in Results. The finalDMSO concentration never exceeded 1 %. For drug treat-ments, 1 ml of the appropriate solution was added to the antherson 2 ml of solid medium. For control experiments 1% (v/v)DMSO in liquid medium was used.

Fluorescence microscopySatisfactory staining and stabilization of microtubules and F-actin were only obtained when the cells were first extracted in adetergent-containing buffer essentially as described by Traas etal. (1987) and Hussey et al. (1987). As reported in those papersdirect fixation with glutaraldehyde or formaldehyde perturbedand fragmented cytoskeletal elements. Anthers were cut in two

and the meiocytes, tetrads or young microspores were squeezedout and suspended in the buffer containing 50mM-Pipes(pH6-9), SmM-EGTA, 5mM-MgSO4, 5% (v/v) DMSO and0-03 % (v/v) Nonidet P40. To this extraction medium, helicase(IBF, France), cellulase Onozuka R-10 (Yakult, Japan) andmacerase (Calbiochem, USA) were added (0-5% of eachenzyme) in order to permeabilize the thick cell wall. Immedi-ately after cell isolation, the suspended meiocytes or tetradswere pipetted into an Eppendorf tube and allowed to settle.

DNA and F-actin staining

After 10 min of wall and cell extraction the supernatant contain-ing cell debris, detergent and enzymes was removed. The pelletwas resuspended in extraction buffer containing 0-5/igml~diamino-2-phenylindole (DAPI) for DNA staining and0-5 ^gml~ rhodaminyl lysine phallotoxine (RLP, a generousgift from Professor Wieland, Heidelberg, FRG) for F-actinstaining (see also Traas et al. 1987; Lloyd & Traas, 1988). Cellsin RLP and DAPI were viewed immediately in an OlympusBH2 fluorescence microscope. In order to restrict fading 2%l,4-diazabicyclo-(2,2,2)octan (DABCO) was added to the cellsuspension.

Microtubule staining

Microtubules were visualized using immunofluorescence. Asthis procedure includes a number of washing steps and longincubations in buffer without detergent, it was necessary to fixthe extracted cells first. For this purpose the pellets of deter-gent- and enzyme-treated cells were resuspended in 1 ml of abuffer containing Pipes (100mM, pH6-9), EGTA (5mM),MgSO4 (SmM) and formaldehyde (8% (w/v), freshly pre-pared). Cells were allowed to settle and washed twice in bufferwithout fixative. After a final wash in water they were allowed toattach to poly-L-lysine (Mr> 300000, Sigma)-coated coverslips.Cells were then prepared for immunofluorescence using amonoclonal anti-tubulin antibody (MAS 077, Sera Lab) and afluorescein isothiocyanate (FITC)-labelled second antibodyfollowing standard procedures. The culture supernatant con-taining the primary antibody (the YL 1/2 anti-yeast tubulinoriginally prepared by Kilmartin et al. (1981)) was diluted 1: 50(v/v) in Pipes (50mM; pH6-9) and 3 % (w/v) bovine serumalbumin (BSA). Citifluor (with glycerol; City University,London) was used as an antifading agent. Preparations wereobserved in an Olympus BH2-RFL microscope with exciterfilters BP-490 (continuous spectrum near 490nm), BP-545(546 nm) and BP-405 (405 and 435 nm) for blue, green andviolet light. They were used with the appropriate barrier filters.

Results

Microtubules during meiosis

The different microtubular arrangements during meiosisare represented in Fig. 1. No differences were foundbetween the two varieties of eggplant. At interphase I,microtubules form a complex network extending fromthe nucleus to the plasma membrane (Fig. 1A,B). Themicrotubular network remains until prometaphasealthough the number of cytoplasmic microtubules gradu-ally decreases (Fig. 1A-E). At the same time the amountof fluorescence surrounding the nuclear envelope in-creases. When the chromosomes are fully condensed theirposition at the inside the nuclear envelope appears to co-localize with the concentrations of tubulin on the outside(Fig. 1C,D). After breakdown of the nuclear envelope

542 jf. A. Traas et al.

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microtubules invade the nucleus (Fig. IF) and an eccen-tric spindle is formed with its pointed poles extendinginto the cortical cytoplasm (Fig. 1G). After chromosomedivision the two daughter nuclei, lying at one side of thecell close to the plasma membrane, move to the two cellpoles (Fig. 1H). At this stage new microtubules alreadystart to grow out from the nucleus and at interphase II adense array radiates out from its surface to the plasmamembrane (Fig. II,J). However, no phragmoplast isformed. As metaphase II approaches, these arrays arepartially depolymerized and microtubules only radiateout from the nuclear poles. At metaphase II the twospindles poles again associate with the cell cortex(Fig. IK). In telophase, microtubules radiate out fromthe daughter nuclei (Fig. 1L,M) but this time a four-wayphragmoplast is formed (Fig. IN). The cell wall isformed centripetally. After cytokinesis microtubules re-form a random interphase array (Fig. 10).

F-actin during meiosis (Fig. 2)In interphase, F-actin forms a network filling the cyto-plasm, extending from the nuclear envelope to the plasmamembrane (Fig. 2A). In prophase I, this network alsoconcentrates around the nucleus (Fig. 2B), but in con-trast to the microtubules this network remains present inthe cytoplasm during meiosis. In metaphase I, actin alsoco-distributes with the spindle microtubules (Fig. 2C).In interphase II, when the microtubules radiate out fromthe two nuclei, a dense web of F-actin invades the tubule-free zone and forms a phragmoplast-like disk(Fig. 2D,E). In many cells this equatorial web is moredense at the cell cortex, this dense zone forming a ringthat indicates the future division site. This structuredisappears completely before the spindles are formed,although actin bundles remain present throughout thecytoplasm. During the second division, F-actin again co-distributes with the spindle microtubules (Fig. 2F). Intelophase II, during phragmoplast formation, microfila-ments are again concentrated in the future division plane,before the microtubules (Fig. 2G). Later they are associ-ated with the radiating microtubules (Fig. 2H). After

cytokinesis, the actin filaments reorganize into a randomarray (Fig. 21).

Drug treatmentsPollen mother cells (PMCs) normally developed intomicrospores in the cultured anthers within 18-24 h. 1 %DMSO did not influence cell division markedly.

The results obtained using the different drugs aresummarized in Table 1. Taxol and phalloidin have bothbeen reported to affect cell division in plant cells (Weer-denburg et al. 1986; Palevitz, 1980). However, in ourhands taxol (1-25 jUM) and phalloidin (5-20/igmP1) didnot influence meiosis significantly. Since cytoskeletalorganization was also unaffected it remains uncertainwhether these drugs effectively reached the meiocytes.Therefore, only the results obtained using CIPC (50 ^Mto 1 raM), colchicine (50 fiM to 1 mM) and cytochalasin Band D (10/iM to 100 /ZM) will be discussed in detail, asthey all had specific effects on meiosis.

The effect of CIPC on meiosis. CIPC is known to affectthe splitting or replication of spindle poles in diverseorganisms (Oliver et al. 1982; Clayton & Lloyd, 1984;review: Gunning & Hardham, 1982) regardless of themorphology of the polar organizers. This drug wastherefore used here in an attempt to disturb the symmetryof meiosis. CIPC disturbed the meiotic divisions, causingthe formation of micronuclei in about 90% of the cells.Usually 7-11 nuclei per cell were formed (Fig. 3C,E),although a minority of the cells had fewer nuclei, thelatter often of unequal size. As judged from DAPIstaining, one or two divisions could occur in the presenceof the drug, although in most cells the multiple nucleiformed during the first division (Fig. 3A). CIPC waseffective at concentrations of 50 jitA and higher. At 0-1 mMvirtually no normal divisions were found.

The effect of CIPC on the cytoskeleton. In interphase,loose networks of short microtubule bundles, having afragmented appearance, were observed (Fig. 3D). At theonset of cell division tubulin staining was concentratedaround the chromosomes (Fig. 3A). Incomplete, ormultipolar spindles were observed at metaphase. After

Table 1. Effects of inhibitors on meiosis

Inhibitor (concn) Effect on cell division Effect on microtubules Effect on F-actin

CIPC

Colchicine (50/iM-l mM)

Cytochalasin B or D(10-2

Cytochalasin B or D(20-100 IM)

Formation of micronuclei

Arrest of developmentAfter 24 h, fragmentation of nuclei

Increased % of abnormal divisionplanes

Arrest of cell development

F-actin networks of short bundleswith fragmented appearance

No rearrangement in phragmoplast

Formation of non- or multipolarspindles

No formation of phragmoplastInterphase arrays of short

disorganized microtubules

Depolymerization of microtubules No direct effect on F-actinnetworks, but actinreorganization is arrested

Microtubule reorganization isperturbed

No spindle or phragmoplastformation

F-actin networks fragmented

Phalloidin and taxol treatments are not summarized here, as these drugs did not seem to affect the cytoskeleton or cell division. Therefore wecould not determine whether the inhibitors reached the meiocytes.

Eggplant cytoskeleton during meiosis 543

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544 J. A. Traas et al.

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Fig. 1. Microtubular organization during meiosis. Bar,10 fim. A-O, are at the same magnification. A,B. Two focalplanes of an interphase cell. Microtubules at the cell cortex(A) and in the cytoplasm (B) form random networks.C,D,E. Cortical (D) and cytoplasmic (E) view of a prophasecell. Cytoplasmic microtubules start to depolymerize andtubulin staining is concentrated around the nucleus. At thisstage chromosomes are attached to the inner membrane of thenuclear envelope (C: DAPI staining). Concentrations oftubulin seem to correspond with the position of thechromosomes (pointers). F. Late prophase. Microtubuleshave invaded the nucleus and surround the chromosomes. Atthis stage spindle fibres grow out to the membrane (out offocus). G. Metaphase I. Spindle with its pointed polesextending into the cell cortex (pointers). Note that the

spindle is eccentric. H. Telophase I. The daughter nucleimove to the two opposite cell poles. Microtubules start toradiate out from the nuclear surface. I J . Interkinesis.Microtubules radiate out from the nuclei many of them runfrom nuclear envelope to plasma membrane. A cortical viewshows that only very short bits or only the ends of themicrotubules are attached to the membrane (J). Between thenuclei there is a microtubule-free zone. K. Metaphase II.Two spindles in parallel planes are formed simultaneously.L,M,N. Telophase Il/cytokinesis. Microtubules radiate outfrom the nuclei to form the phragmoplast. In L the cell wasslightly squashed and flattened, which makes it easier tointerpret microtubular organization at this stage. In N thenewly formed wall that forms centripetally is visible. O. Earlytetrad with random microtubular arrays.

Fig. 2. F-actin during meiosis,DAPI staining not shown. Bars:10 lira. A. Random interphasenetwork. B. Network in of cellin prophase. Note increasedfluorescence around the nucleus.C. Metaphase I. Actin isassociated with the spindle andforms a network throughout thecytoplasm. D. Interkinesis.Actin is present throughout thecytoplasm, although it isconcentrated between thenuclei, indicating the futureplane of division. Pointers markthe positions of the nuclei.

E. Interkinesis. Lowermagnification of the actin diskin different cells. RLP stainingis brighter at the cell periphery(arrows), showing that in manycells the transcellular network ismore dense at the cell cortex,this dense zone has theappearance of a ring.F. Telophase II. Thechromosomes have just movedto the spindle poles. Note thepresence of F-actin in thespindle. The F-actin disk seenin interkinesis has completelydisappeared.G,H. Phragmoplast formation.First F-actin becomesconcentrated in the divisionplanes (G). Later it is moreintimately associated with themicrotubules (H). I. Earlytetrad with randommicrofilament networks.

cell division there was no phragmoplast formation and anirregular network of short bundles was re-formed, inter-connecting the daughter nuclei. The F-actin system wassimilarly affected. Often short filament bundles wereobserved, giving the networks a fragmented appearance.

These networks remained present during cell divisionand no obvious changes in their organization could beobserved (Fig. 3F,G).

The effect of colchicine on meiosis. Colchicine at con-centrations of SOfiM and higher blocked cell development

Eggplant cytoskeleton during meiosis 545

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Fig. 3. CIPC treatment. Bars: 10/im. A,B- Microtubule (A) and DNA (B) staining of a metaphase cell. A multi- or non-polarspindle-like structure has been formed. C. DAPI staining of CIPC-treated cells showing the presence of micronuclei.D,E. Tubulin (D) and DNA (E) staining of a cell after division. A random network of short microtubules interconnects themicronuclei. F,G. Actin (F) and DNA (G) staining of a cell after cell division. At least four nuclei have formed, but there is nophragmoplast.

Fig. 4. Colchicine treatment. Bar, 10^m. A-D are at the same magnification. A,B. Tubulin (A) and DNA (B) staining of acell in interkinesis. All microtubules have disappeared and only an irregular, tubulin-containing structure remains. C,D. Actinand DNA-staining of a (pro)metaphase cell. F-actin network is present, but there is no concentration of actin around thechromosomes.

and usually no microspores were formed in the presenceof the drug. Longer treatments with colchicine (36-48 h)caused the fragmentation of a number of nuclei that wereprobably in division. However, in contrast to CIPC-treated cells, micronuclei were never observed.

The effect of colchicine on the cytoskeleton. Colchicinecaused the depolymerization of the microtubular array inmost cells. However, irregular, tubulin-containing struc-tures remained present (Fig. 4A,B). In a number of cells

fragmented networks could still be observed. F-actinnetworks were present at all stages although no spindle orphragmoplast-like structures were observed (Fig. 4C,D).After 24—48 h the networks became increasingly frag-mented.

The effect of cytochalasin B and D on meiosis. Atconcentrations higher than 20 ̂ M cytochalasin B or Dcaused arrested cell development. However, at lowerconcentrations (10/iM) the formation of abnormal div-

546 Jf. A. Tracts et al.

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eftFig. 5. Cytochalasin B treatment (30fiM). Bars: lO^Jm. A,B. Tubulin and DNA staining in a prophase cell. The chromosomesstart to condense (B) and niicrotubule organization seems normal. C,D. Tubulin and DNA in a metaphase I cell. No spindle ispresent. Microtubules run more or less parallel to each other from one pole of the cell to the other. They seem to grow out fromputative nucleating centres at the cortex. E. Microtubules in a cell after cell division. There is no phragmoplast formation.F. F-actin staining, showing that the bundles are fragmented. G,H. DAPI staining of a number of cells with abnormal divisionplanes formed in the presence of cytochalasin B at 10[1M.

ision planes and of dyads was observed in about 5 % ofthe cells (Fig. 5G,H). This was higher than the 1%(mostly dyads) usually observed in controls.

The effect of cytochalasin B and D on the cytoskeleton.Cytochalasin, at concentrations of 10jJM and higher,disturbed the reorganization of the microtubular systemduring meiosis. In pre-meiotic cells and cells in prophasenormal microtubular networks were observed (Fig. 5A).In dividing cells, however, spindles were usually absent(Fig. 5C). Phragmoplasts were never observed(Fig. 5E). Instead, dividing cells usually possessed anumber of thick microtubule bundles that in metaphase/telophase were orientated more or less parallel to eachother (Fig. 5C). The F-actin bundles were always highlyfragmented (Fig. 5F).

Discussion

The way in which plant cell division is coordinated is oneof the major aspects of plant morphogenesis. Because ofthis, much attention has been paid to the cytoskeleton,since it plays a key role in establishing polarity and indetermining the division planes with great precision(reviews: Lloyd, 1987; Traas, 1989). Descriptions of themicrotubular component of the cytoskeleton have domi-nated the literature but recent findings show that F-actinplays an important part in spatial control (Traas et al.

1987; Schmit & Lambert, 1987; Kakimoto & Shibaoka,1987; Lloyd & Traas, 1988). From these studies itappears that F-actin could provide a cytoplasmic frame-work that supports and organizes the cytoplasm duringcell division (for discussion, see also Lloyd, 1988). Asyet, the exact role of F-actin during the different stages ofdivision remains unclear and more information is neededto complete existing models on cytoskeletal functioning.Here, we have studied meiosis partly as an essential stepin pollen development, but even more importantly as atypical example of cell division under strict geometricalcontrol. The PMCs are apparently unpolarized and thetwo meiotic divisions involve the establishment of spindlepolarity and the subsequent determination of two div-ision planes at right angles to each other. Meiosistherefore provides an interesting tool for studying generalaspects of the coordination of cell division.

The formation of the spindle: the establishment ofpolarityIn pre-meiotic interphase both microtubules and actinfilaments form random networks throughout the cyto-plasm. During prophase both filamentous systems be-come more dense around the nucleus (see also VanLammeren et al. 1985; Sheldon & Dickinson, 1986;Hogan, 1987). At this stage the condensed chromosomesmove to the inner face of the nuclear envelope and it has

Eggplant cytoskeleton during meiosis 547

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been suggested that microtubules might function inchromosome pairing (Sheldon & Dickinson, 1986).

The PMCs do not show any obvious polarity until theestablishment of the spindle. This involves the concen-tration of microtubule nucleation sites (MTNS) at thetwo poles of the nucleus. This process is disturbed byCIPC, which causes the splitting or replication of spindlepole bodies of various structures. Similar results havebeen reported for animal, algal, monocotyledonous anddicotyledonous cells, suggesting that a phylogenetically'universal' mechanism is affected (e.g. see Oliver et al.1978; Clayton & Lloyd, 1984; Gunning & Hardham,1982, for review). Our results do not support a role for F-actin in determining spindle polarity: even in the pres-ence of high concentrations of cytochalasin the micro-tubular system retains a clear polarity. Interestingly,similar results have been obtained with animal cells. Forinstance, during early embryogenesis of Caenorhabditiselegans centrosomal movements along the nuclear surfaceare perturbed by anti-microtubule drugs but not bycytochalasin D (Hyman & White, 1987; see also DeBrabander et al. 1986).

As soon as the MTNS are concentrated at the two polesof the nucleus a spindle is formed that has pointed poles,in contrast to the barrel-shaped spindles usually observedin higher plant cells. We have observed that these polesare always associated with the cell cortex and it is possiblethat membrane-microtubule interactions are involved inmaintaining the position of the spindle.

RLP stains filaments and bundles within and aroundthe meiotic spindles of eggplant. This is not in agreementwith Sheldon & Hawes (1988), who could not localize F-actin in association with the spindle microtubules andconcluded that both cytoskeletal systems act indepen-dently during metaphase-telophase. Our results, how-ever, are supported by a number of recent reports onmitotic plant cells showing that actin does associate withmicrotubules throughout division (Traas et al. 1987;Schmit et al. 1985; Kakimoto & Shibaoka, 1987) and theconflicting evidence could simply reflect differences instability of the mitotic actin system under differentpreparative conditions.

It has been proposed that actin could function inchromatid separation (Forer et al. 1979; Cande et al.1977; Seagull et al. 1987). However, this is not firmlyestablished and it appears from in vitro experiments thatmicrotubule depolymerization is sufficient to explainchromosome movement without the need for actin as aforce generator (e.g. see Koshland et al. 1988; Gorbsky etal. 1988). Our results indicate a completely different rolefor actin in mitosis: cytochalasins perturb spindle forma-tion and therefore actin could be involved in spindleformation or organization, i.e. in the reorganization ofthe microtubular array during division. Likewise,Kobayashi et al. (1987) have proposed that actin fila-ments are actively involved in the re-alignment of micro-tubules during differentiation of tracheary elements ofZinnia. In these cells microtubules and actin filamentsare co-parallel. The microtubules switch their orientationfrom longitudinal to transverse during re-differentiation,but cytochalasin B inhibits microtubular re-orientation.

Previous reports have shown that cytochalasins do notaffect spindle formation or functioning in a number ofplant cells (e.g. see Schmit & Lambert, 1987; Lloyd &Traas, 1988; for discussion, see also Lloyd, 1988). Oneshould realize, however, that part of the F-actin popu-lation is resistant to cytochalasin treatments: in carrotcells, for example, cytochalasin B and D are unable todepolymerize spindle-associated actin (Lloyd & Traas,1988). Therefore, the contrasting results obtained withvarious cell types could be explained in terms of differ-ences in sensitivity to cytochalasins.

Metaphase I-interkinesis: the establishment of thedivisio)i planesAt telophase the daughter nuclei start to move to the twoopposite poles of the cell. They remain interconnected bybundles of microtubules, which could function in themigration by pushing the nuclei apart. During interkin-esis microtubules still radiate out between the two nuclei.The F-actin network that is still present throughout thecytoplasm starts to concentrate in this zone and forms adisk that divides the cytoplasm in two and indicates thefuture division plane. This process closely resemblesearly steps in phragmoplast formation in higher plantcells and it has been proposed that the equatorial actinfunctions in guiding the outgrowth of the new cell wall(Schmit & Lambert, 1987; Lloyd & Traas, 1988).However, PMCs of dicotyledons do not form a cross-wallat this stage and therefore the equatorial actin system inmeiocytes must have a role other than helping to form adividing wall. Different observations suggest that a rolefor F-actin may exist in the establishment of the futuredivision plane. (1) In a variety of plants, organellesmigrate to the equatorial region after the first meioticdivision, thus marking the future division site (Brown &Lemmon, 1987, and references therein). The cytoplas-mic disk of F-actin could obviously function in such aprocess, re-distributing the cytoplasm in preparation forcytokinesis. (2) The equatorial F-actin also extends to theplasma membrane where the network seems to be moredense. The disk of actin is therefore continuous across thecell from one side to the other. As such it wouldinevitably 'memorize' the division plane at the cortex. Asimilar role has been proposed for the microtubularpreprophase band (PPB), which accurately marks thefuture site of division at the cortex of polarized cells(Lloyd, 1987; Traas, 1988, for reviews). At one time itwas thought that microtubules within the PPB were aloneresponsible for marking the division site. Now it is knownthat F-actin also occurs within that band (Palevitz, 1987;Traas et al. 1987) as well as in the plane of the futuredivision site (Lloyd & Traas, 1988). It is important toappreciate that there is no PPB of microtubules inmeiocytes to mark the division site. Perhaps the actualplane of division in meiocytes is not initially fixed as it isin somatic cells. However, it is clear that the actinnetwork alone is sufficient to form a raft that bi-lateralizesthe daughter nuclei and delineates the first division plane,which is to be followed by another at right angles. Theabsence of a PPB accentuates the involvement of F-actinin these division processes. In cytokinesis in animal cells

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F-actin is concentrated in the cortical division site (thecontractile ring) before and during furrowing. It has beenargued that astral arrays of microtubules radiating out tothe equatorial region determine the division planes (re-view: Mabuchi, 1986). In plant meiocytes the micro-tubules radiating out from the daughter nuclei to thefuture division site could function in a similar way,perhaps by transporting or guiding the actin network tothe equator.

The second meiotic division: spindle and phragmoplastformationDuring prophase II, microtubules extending freely intothe cytoplasm between the daughter nuclei start todepolymerize, whereas those between the nuclear envel-ope and the plasma membrane elongate. As in prophaseI, interactions with the membrane could help to stabilizecertain classes of microtubules, which then could partici-pate in spindle formation/alignment.

After nuclear division, the microtubules again radiateout from the nuclear envelope in telophase II. F-actininvades the future division planes as it did after the firstdivision, but this time it remains there while the phrag-moplast develops. Comparing the two meiotic divisions,it seems that the concentration of actin in the divisionplanes has two distinct roles. First, the F-actin disk couldbe involved in re-distributing the cytoplasm in prep-aration for cytokinesis as was suggested above. Such aprocess would be independent of the formation of a cross-wall and constitutes a distinct step during meiosis.During the second division, F-actin could initially func-tion in the same way but, in remaining, would have theadditional role of guiding the outgrowth of the phragmo-plast as proposed for other plant cells (Schmit & Lam-bert, 1987; Lloyd & Traas, 1988). This suggestion is alsosupported by the fact that cytochalasin treatments per-turb the alignment of division planes and formation of thephragmoplast. It is an interesting aspect of meiosis thatapparently the cell is able to uncouple these otherwiseclosely linked steps, thus preventing phragmoplast for-mation.

In summary, our results suggest that the microtubularsystem primarily acts in the establishment of spindle andcell polarity and that F-actin is involved in the establish-ment and 'memorization' of the division planes and in theorganization of the cytoplasm during meiosis. Moreover,reorganization of the cytoskeleton greatly depends on theinteraction between the two systems. These seem to begeneral features not only of plant cell division but, asdiscussed above, also of animal cell division.

It is tempting to describe processes like cell divisionentirely in terms of microtubule dynamics. Yet, a numberof observations including our own also stress the import-ance of microtubule—actin interactions in plant celldivision and it is likely that the cytoskeleton as a whole isinvolved in the spatial control of cell division.

Meiosis offers an important tool for analysing thecoordination of cell division. Populations of meiocytescan be obtained in which every cell is in division. Becauseof their synchronous development, such cells will be

important in the next phase in which the molecular basisof meiosis will be studied.

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(Received J September I9SS - Accepted, in revised form,4 January 1989)

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