The Organization of the Cytoskeleton During Meiosis in Eggplant {Solanum Melongena (L.)): Microtubules and F-Actin Are Both Necessary for Coordinated Meiotic Division

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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, concentrating actin in that plane (cf. the proposed male meiosis in higher plants provides striking role of asters in positioning the contractile ring in examples of both division control and spatial pro- animal cells). gramming. (4) That this concentration of F-actin in the To investigate these processes we have stained division plane may be involved in preparing the microtubules and actin filaments during male mei- cytoplasm for cytokinesis and in memorizing the osis in the eggplant. Our results indicate the follow- division plane (much as the preprophase band ing. observed in polarized tissues does). (1) That microtubules and their nucleation sites (5) That phragmoplast formation is a two-step are involved in the establishment of polarity; this is process. No phragmoplast forms after metaphase I, supported by our observation that the drug CIPC but a four-way phragmoplast forms after meta- affects spindle polarity. phase II, indicating that mitosis and cytokinesis are (2) That actin microfilaments are involved in not obligatorily coupled. spindle formation and integrity, but not in the These studies demonstrate that actin and micro- establishment of polarity: cytochalasin B and D tubules are jointly involved in the spatial coordi- affect the organization of the spindle microtubules, nation of the division process. but not their polarized distribution. (3) That microtubules radiating from the daughter nuclei at the cell poles during interkinesis Key words: cytoskeleton, eggplant, F-actin, microtubules, probably establish the future division plane by meiosis.

Introduction somatic mitosis, microtubules function in nuclear division, whereas in cytokinesis F-actin accompanies micro- Meiosis has been studied mainly from the 'chromosomal' tubules in forming the phragmoplast. However, a num- point of view and much is known about the behaviour and ber of important questions remain to be answered, configuration of the chromosomes (reviews: John & especially concerning the establishment and maintenance Lewis, 1965; Sybenga, 1975; Dickinson, 1988). Yet, the of the well-defined division planes by which four haploid cytoplasmic mechanisms that control meiosis, determine microspores are separated by two meiotic divisions. chromosome pairing, define with great precision the Recent findings have established that F-actin plays an division planes and ensure the distribution of cytoplasm important role in determining the division plane of between the daughter cells are of equal importance but somatic cells (Traas et al. 1987; Lloyd & Traas, 1988; remain poorly understood. Because of this, some atten- Lloyd, 1988). Actin filaments had been thought to be tion has been paid to the role of the cytoskeleton during absent during mitosis, but by avoiding aldehyde fixation male meiosis in higher plants and changes in microtubu- (by detergent extraction or electroporation) it was dis- lar and, to a lesser extent, F-actin arrays have been covered that a network of actin persists throughout described for a number of species (Van Lammeren et al. mitosis and cytokinesis. The actin envelopes the nucleus 1985; Sheldon & Dickinson, 1986; Hogan, 1987; Sheldon and, by transvacuolar filaments, connects that organelle & Hawes, 1988). Such studies show that, as in normal to the cortex. In somatic mitosis the division plane is

Journal of Cell Science 92, 541-550 (1989) Printed in Great Britain © The Company of Biologists Limited 1989 541 predicted by the formation of a preprophase band (PPB) and the meiocytes, tetrads or young microspores were squeezed of microtubules, but it is now apparent that actin out and suspended in the buffer containing 50mM-Pipes filaments also form a cortical band, so that the PPB is no (pH6-9), SmM-EGTA, 5mM-MgSO4, 5% (v/v) DMSO and longer considered to consist of microtubules exclusively. 0-03 % (v/v) Nonidet P40. To this extraction medium, helicase (IBF, France), cellulase Onozuka R-10 (Yakult, Japan) and Even though the PPB microtubules disappear by meta- macerase (Calbiochem, USA) were added (0-5% of each phase, the radial nucleus-associated actin strands remain enzyme) in order to permeabilize the thick cell wall. Immedi- in the division plane, providing a memory of the division ately after cell isolation, the suspended meiocytes or tetrads site and helping to guide the cytokinetic apparatus out were pipetted into an Eppendorf tube and allowed to settle. along the pre-determined path. The significance of these observations is that actin and microtubules combine to DNA and F-actin staining set up the division plane. In view of this, we have re- After 10 min of wall and cell extraction the supernatant contain- examined meiotic division, which differs from mitosis in ing cell debris, detergent and enzymes was removed. The pellet several important aspects: there is no PPB, for example, was resuspended in extraction buffer containing 0-5/igml~ and there is no known basis for the four-square position- diamino-2-phenylindole (DAPI) for DNA staining and ing of the haploid microspores produced by two success- 0-5 ^gml~ rhodaminyl lysine phallotoxine (RLP, a generous ive meiotic divisions. However, in this paper we now gift from Professor Wieland, Heidelberg, FRG) for F-actin report the presence of an equatorial system of F-actin that staining (see also Traas et al. 1987; Lloyd & Traas, 1988). Cells predicts the first division plane. As in mitosis, micro- in RLP and DAPI were viewed immediately in an Olympus tubule-actin interactions seem to be essential for the BH2 fluorescence microscope. In order to restrict fading 2% l,4-diazabicyclo-(2,2,2)octan (DABCO) was added to the cell spatial control of male meiotic division. suspension.

Microtubule staining Materials and methods Microtubules were visualized using immunofluorescence. As this procedure includes a number of washing steps and long Plant material incubations in buffer without detergent, it was necessary to fix Two varieties of Solatium melongena (L.) were used for the the extracted cells first. For this purpose the pellets of deter- experiments: Ronde de Valence and Doutga. Plants were grown gent- and enzyme-treated cells were resuspended in 1 ml of a under greenhouse conditions. Young buds were cut from the buffer containing Pipes (100mM, pH6-9), EGTA (5mM), plants and the stage of meiocyte development was determined in MgSO4 (SmM) and formaldehyde (8% (w/v), freshly pre- one anther of each bud. For this purpose anthers were squashed pared). Cells were allowed to settle and washed twice in buffer in water and examined in an Olympus BH2 microscope without fixative. After a final wash in water they were allowed to equipped with Nomarski optics. This gives a satisfactory attach to poly-L-lysine (Mr> 300000, Sigma)-coated coverslips. estimate of the stage of the four or five remaining anthers as Cells were then prepared for immunofluorescence using a microspore formation is highly synchronized within each bud. monoclonal anti-tubulin antibody (MAS 077, Sera Lab) and a Only anthers containing meiocytes at a stage prior to division fluorescein isothiocyanate (FITC)-labelled second antibody were used. This stage is characterized by the formation of the following standard procedures. The culture supernatant con- thick callosic wall. taining the primary antibody (the YL 1/2 anti-yeast tubulin originally prepared by Kilmartin et al. (1981)) was diluted 1: 50 Anther culture and drug treatments (v/v) in Pipes (50mM; pH6-9) and 3% (w/v) bovine serum Intact anthers were removed from the buds and put in 3-5 cm albumin (BSA). Citifluor (with glycerol; City University, Petri dishes containing 2 ml solid 'T' medium (pH 59) without London) was used as an antifading agent. Preparations were hormones (concentrations of macro- and micronutrients, vit- observed in an Olympus BH2-RFL microscope with exciter amins, sucrose and agar were as described by Chambonnet & filters BP-490 (continuous spectrum near 490nm), BP-545 Dumas de Vaulx, 1983). Under these conditions meiosis (546 nm) and BP-405 (405 and 435 nm) for blue, green and proceeds normally within 18 h of culture. The following drugs violet light. They were used with the appropriate barrier filters. were used: chloroisopropylphenyl carbamate (CIPC) (Sigma), colchicine (Prolabo), taxol (a generous gift from NCI, Beth- esda), cytochalasin B and D (Sigma), and phalloidin (Sigma). Results The drugs were first solubilized in dimethyl sulphoxide (DMSO) and subsequently diluted in liquid T medium. Microtubules during meiosis Colchicine was also solubilized in water. The different concen- The different microtubular arrangements during meiosis trations used for each drug are given in Results. The final are represented in Fig. 1. No differences were found DMSO concentration never exceeded 1 %. For drug treat- between the two varieties of eggplant. At interphase I, ments, 1 ml of the appropriate solution was added to the anthers on 2 ml of solid medium. For control experiments 1% (v/v) microtubules form a complex network extending from DMSO in liquid medium was used. the nucleus to the plasma membrane (Fig. 1A,B). The microtubular network remains until prometaphase although the number of cytoplasmic microtubules gradu- Fluorescence microscopy ally decreases (Fig. 1A-E). At the same time the amount Satisfactory staining and stabilization of microtubules and F- of fluorescence surrounding the nuclear envelope in- actin were only obtained when the cells were first extracted in a creases. When the chromosomes are fully condensed their detergent-containing buffer essentially as described by Traas et al. (1987) and Hussey et al. (1987). As reported in those papers position at the inside the nuclear envelope appears to co- direct fixation with glutaraldehyde or formaldehyde perturbed localize with the concentrations of tubulin on the outside and fragmented cytoskeletal elements. Anthers were cut in two (Fig. 1C,D). After breakdown of the nuclear envelope

542 jf. A. Traas et al. microtubules invade the nucleus (Fig. IF) and an eccen- cytokinesis, the actin filaments reorganize into a random tric spindle is formed with its pointed poles extending array (Fig. 21). into the cortical cytoplasm (Fig. 1G). After chromosome division the two daughter nuclei, lying at one side of the Drug treatments cell close to the plasma membrane, move to the two cell Pollen mother cells (PMCs) normally developed into poles (Fig. 1H). At this stage new microtubules already microspores in the cultured anthers within 18-24 h. 1 % start to grow out from the nucleus and at interphase II a DMSO did not influence cell division markedly. dense array radiates out from its surface to the plasma The results obtained using the different drugs are membrane (Fig. II,J). However, no phragmoplast is summarized in Table 1. Taxol and phalloidin have both formed. As metaphase II approaches, these arrays are been reported to affect cell division in plant cells (Weer- partially depolymerized and microtubules only radiate denburg et al. 1986; Palevitz, 1980). However, in our out from the nuclear poles. At metaphase II the two hands taxol (1-25 jUM) and phalloidin (5-20/igmP1) did spindles poles again associate with the cell cortex not influence meiosis significantly. Since cytoskeletal (Fig. IK). In telophase, microtubules radiate out from organization was also unaffected it remains uncertain the daughter nuclei (Fig. 1L,M) but this time a four-way whether these drugs effectively reached the meiocytes. phragmoplast is formed (Fig. IN). The cell wall is Therefore, only the results obtained using CIPC (50 ^M formed centripetally. After cytokinesis microtubules re- to 1 raM), colchicine (50 fiM to 1 mM) and cytochalasin B form a random interphase array (Fig. 10). and D (10/iM to 100 /ZM) will be discussed in detail, as they all had specific effects on meiosis. F-actin during meiosis (Fig. 2) The effect of CIPC on meiosis. CIPC is known to affect In interphase, F-actin forms a network filling the cyto- the splitting or replication of spindle poles in diverse plasm, extending from the nuclear envelope to the plasma organisms (Oliver et al. 1982; Clayton & Lloyd, 1984; membrane (Fig. 2A). In prophase I, this network also review: Gunning & Hardham, 1982) regardless of the concentrates around the nucleus (Fig. 2B), but in con- morphology of the polar organizers. This drug was trast to the microtubules this network remains present in therefore used here in an attempt to disturb the symmetry the cytoplasm during meiosis. In metaphase I, actin also of meiosis. CIPC disturbed the meiotic divisions, causing co-distributes with the spindle microtubules (Fig. 2C). the formation of micronuclei in about 90% of the cells. In interphase II, when the microtubules radiate out from Usually 7-11 nuclei per cell were formed (Fig. 3C,E), the two nuclei, a dense web of F-actin invades the tubule- although a minority of the cells had fewer nuclei, the free zone and forms a phragmoplast-like disk latter often of unequal size. As judged from DAPI (Fig. 2D,E). In many cells this equatorial web is more staining, one or two divisions could occur in the presence dense at the cell cortex, this dense zone forming a ring of the drug, although in most cells the multiple nuclei that indicates the future division site. This structure formed during the first division (Fig. 3A). CIPC was disappears completely before the spindles are formed, effective at concentrations of 50 jitA and higher. At 0-1 mM although actin bundles remain present throughout the virtually no normal divisions were found. cytoplasm. During the second division, F-actin again co- The effect of CIPC on the cytoskeleton. In interphase, distributes with the spindle microtubules (Fig. 2F). In loose networks of short microtubule bundles, having a telophase II, during phragmoplast formation, microfila- fragmented appearance, were observed (Fig. 3D). At the ments are again concentrated in the future division plane, onset of cell division tubulin staining was concentrated before the microtubules (Fig. 2G). Later they are associ- around the chromosomes (Fig. 3A). Incomplete, or ated with the radiating microtubules (Fig. 2H). After multipolar 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 Formation of micronuclei Formation of non- or multipolar F-actin networks of short bundles spindles with fragmented appearance No formation of phragmoplast No rearrangement in phragmoplast Interphase arrays of short disorganized microtubules Colchicine (50/iM-l mM) Arrest of development Depolymerization of microtubules No direct effect on F-actin After 24 h, fragmentation of nuclei networks, but actin reorganization is arrested Cytochalasin B or D Increased % of abnormal division (10-2 planes Cytochalasin B or D Arrest of cell development Microtubule reorganization is F-actin networks fragmented (20-100 IM) perturbed No spindle or phragmoplast formation

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

Eggplant cytoskeleton during meiosis 543 544 J. A. Traas et al. Fig. 1. Microtubular organization during meiosis. Bar, spindle is eccentric. H. Telophase I. The daughter nuclei 10 fim. A-O, are at the same magnification. A,B. Two focal move to the two opposite cell poles. Microtubules start to planes of an interphase cell. Microtubules at the cell cortex radiate out from the nuclear surface. IJ. Interkinesis. (A) and in the cytoplasm (B) form random networks. Microtubules radiate out from the nuclei many of them run C,D,E. Cortical (D) and cytoplasmic (E) view of a prophase from nuclear envelope to plasma membrane. A cortical view cell. Cytoplasmic microtubules start to depolymerize and shows that only very short bits or only the ends of the tubulin staining is concentrated around the nucleus. At this microtubules are attached to the membrane (J). Between the stage chromosomes are attached to the inner membrane of the nuclei there is a microtubule-free zone. K. Metaphase II. nuclear envelope (C: DAPI staining). Concentrations of Two spindles in parallel planes are formed simultaneously. tubulin seem to correspond with the position of the L,M,N. Telophase Il/cytokinesis. Microtubules radiate out chromosomes (pointers). F. Late prophase. Microtubules from the nuclei to form the phragmoplast. In L the cell was have invaded the nucleus and surround the chromosomes. At slightly squashed and flattened, which makes it easier to this stage spindle fibres grow out to the membrane (out of interpret microtubular organization at this stage. In N the focus). G. Metaphase I. Spindle with its pointed poles newly formed wall that forms centripetally is visible. O. Early extending into the cell cortex (pointers). Note that the tetrad with random microtubular arrays.

Fig. 2. F-actin during meiosis, DAPI staining not shown. Bars: 10 lira. A. Random interphase network. B. Network in of cell in prophase. Note increased fluorescence around the nucleus. C. Metaphase I. Actin is associated with the spindle and forms a network throughout the cytoplasm. D. Interkinesis. Actin is present throughout the cytoplasm, although it is concentrated between the nuclei, indicating the future plane of division. Pointers mark the positions of the nuclei. E. Interkinesis. Lower magnification of the actin disk in different cells. RLP staining is brighter at the cell periphery (arrows), showing that in many cells the transcellular network is more dense at the cell cortex, this dense zone has the appearance of a ring. F. Telophase II. The chromosomes have just moved to the spindle poles. Note the presence of F-actin in the spindle. The F-actin disk seen in interkinesis has completely disappeared. G,H. Phragmoplast formation. First F-actin becomes concentrated in the division planes (G). Later it is more intimately associated with the microtubules (H). I. Early tetrad with random microfilament networks. cell division there was no phragmoplast formation and an These networks remained present during cell division irregular network of short bundles was re-formed, inter- and no obvious changes in their organization could be connecting the daughter nuclei. The F-actin system was observed (Fig. 3F,G). similarly affected. Often short filament bundles were The effect of colchicine on meiosis. Colchicine at con- observed, giving the networks a fragmented appearance. centrations of SOfiM and higher blocked cell development

Eggplant cytoskeleton during meiosis 545 Fig. 3. CIPC treatment. Bars: 10/im. A,B- Microtubule (A) and DNA (B) staining of a metaphase cell. A multi- or non-polar spindle-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 the micronuclei. F,G. Actin (F) and DNA (G) staining of a cell after cell division. At least four nuclei have formed, but there is no phragmoplast.

Fig. 4. Colchicine treatment. Bar, 10^m. A-D are at the same magnification. A,B. Tubulin (A) and DNA (B) staining of a cell in interkinesis. All microtubules have disappeared and only an irregular, tubulin-containing structure remains. C,D. Actin and DNA-staining of a (pro)metaphase cell. F-actin network is present, but there is no concentration of actin around the chromosomes. and usually no microspores were formed in the presence fragmented networks could still be observed. F-actin of the drug. Longer treatments with colchicine (36-48 h) networks were present at all stages although no spindle or caused the fragmentation of a number of nuclei that were phragmoplast-like structures were observed (Fig. 4C,D). probably in division. However, in contrast to CIPC- After 24—48 h the networks became increasingly frag- treated cells, micronuclei were never observed. mented. The effect of colchicine on the cytoskeleton. Colchicine The effect of cytochalasin B and D on meiosis. At caused the depolymerization of the microtubular array in concentrations higher than 20 ^M cytochalasin B or D most cells. However, irregular, tubulin-containing struc- caused arrested cell development. However, at lower tures remained present (Fig. 4A,B). In a number of cells concentrations (10/iM) the formation of abnormal div-

546 Jf. A. Tracts et al. eft

Fig. 5. Cytochalasin B treatment (30fiM). Bars: lO^Jm. A,B. Tubulin and DNA staining in a prophase cell. The chromosomes start to condense (B) and niicrotubule organization seems normal. C,D. Tubulin and DNA in a metaphase I cell. No spindle is present. Microtubules run more or less parallel to each other from one pole of the cell to the other. They seem to grow out from putative 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 division planes formed in the presence of cytochalasin B at 10[1M. ision planes and of dyads was observed in about 5 % of 1987; Schmit & Lambert, 1987; Kakimoto & Shibaoka, the cells (Fig. 5G,H). This was higher than the 1% 1987; Lloyd & Traas, 1988). From these studies it (mostly dyads) usually observed in controls. appears that F-actin could provide a cytoplasmic frame- The effect of cytochalasin B and D on the cytoskeleton. work that supports and organizes the cytoplasm during Cytochalasin, at concentrations of 10jJM and higher, cell division (for discussion, see also Lloyd, 1988). As disturbed the reorganization of the microtubular system yet, the exact role of F-actin during the different stages of during meiosis. In pre-meiotic cells and cells in prophase division remains unclear and more information is needed normal microtubular networks were observed (Fig. 5A). to complete existing models on cytoskeletal functioning. In dividing cells, however, spindles were usually absent Here, we have studied meiosis partly as an essential step (Fig. 5C). Phragmoplasts were never observed in pollen development, but even more importantly as a (Fig. 5E). Instead, dividing cells usually possessed a typical example of cell division under strict geometrical number of thick microtubule bundles that in metaphase/ control. The PMCs are apparently unpolarized and the telophase were orientated more or less parallel to each two meiotic divisions involve the establishment of spindle other (Fig. 5C). The F-actin bundles were always highly polarity and the subsequent determination of two div- fragmented (Fig. 5F). ision planes at right angles to each other. Meiosis therefore provides an interesting tool for studying general aspects of the coordination of cell division. Discussion

The way in which plant cell division is coordinated is one The formation of the spindle: the establishment of of the major aspects of plant morphogenesis. Because of polarity this, much attention has been paid to the cytoskeleton, In pre-meiotic interphase both microtubules and actin since it plays a key role in establishing polarity and in filaments form random networks throughout the cyto- determining the division planes with great precision plasm. During prophase both filamentous systems be- (reviews: Lloyd, 1987; Traas, 1989). Descriptions of the come more dense around the nucleus (see also Van microtubular component of the cytoskeleton have domi- Lammeren et al. 1985; Sheldon & Dickinson, 1986; nated the literature but recent findings show that F-actin Hogan, 1987). At this stage the condensed chromosomes plays an important part in spatial control (Traas et al. move to the inner face of the nuclear envelope and it has

Eggplant cytoskeleton during meiosis 547 been suggested that microtubules might function in Previous reports have shown that cytochalasins do not chromosome pairing (Sheldon & Dickinson, 1986). affect spindle formation or functioning in a number of The PMCs do not show any obvious polarity until the plant cells (e.g. see Schmit & Lambert, 1987; Lloyd & establishment of the spindle. This involves the concen- Traas, 1988; for discussion, see also Lloyd, 1988). One tration of microtubule nucleation sites (MTNS) at the should realize, however, that part of the F-actin popu- two poles of the nucleus. This process is disturbed by lation is resistant to cytochalasin treatments: in carrot CIPC, which causes the splitting or replication of spindle cells, for example, cytochalasin B and D are unable to pole bodies of various structures. Similar results have depolymerize spindle-associated actin (Lloyd & Traas, been reported for animal, algal, monocotyledonous and 1988). Therefore, the contrasting results obtained with dicotyledonous cells, suggesting that a phylogenetically various cell types could be explained in terms of differ- 'universal' mechanism is affected (e.g. see Oliver et al. ences in sensitivity to cytochalasins. 1978; Clayton & Lloyd, 1984; Gunning & Hardham, 1982, for review). Our results do not support a role for F- Metaphase I-interkinesis: the establishment of the actin in determining spindle polarity: even in the pres- divisio)i planes ence of high concentrations of cytochalasin the micro- At telophase the daughter nuclei start to move to the two tubular system retains a clear polarity. Interestingly, opposite poles of the cell. They remain interconnected by similar results have been obtained with animal cells. For bundles of microtubules, which could function in the instance, during early embryogenesis of Caenorhabditis migration by pushing the nuclei apart. During interkin- elegans centrosomal movements along the nuclear surface esis microtubules still radiate out between the two nuclei. are perturbed by anti-microtubule drugs but not by The F-actin network that is still present throughout the cytochalasin D (Hyman & White, 1987; see also De cytoplasm starts to concentrate in this zone and forms a Brabander et al. 1986). disk that divides the cytoplasm in two and indicates the As soon as the MTNS are concentrated at the two poles future division plane. This process closely resembles of the nucleus a spindle is formed that has pointed poles, early steps in phragmoplast formation in higher plant in contrast to the barrel-shaped spindles usually observed cells and it has been proposed that the equatorial actin in higher plant cells. We have observed that these poles functions in guiding the outgrowth of the new cell wall are always associated with the cell cortex and it is possible (Schmit & Lambert, 1987; Lloyd & Traas, 1988). that membrane-microtubule interactions are involved in However, PMCs of dicotyledons do not form a cross-wall maintaining the position of the spindle. at this stage and therefore the equatorial actin system in RLP stains filaments and bundles within and around meiocytes must have a role other than helping to form a the meiotic spindles of eggplant. This is not in agreement dividing wall. Different observations suggest that a role with Sheldon & Hawes (1988), who could not localize F- for F-actin may exist in the establishment of the future actin in association with the spindle microtubules and division plane. (1) In a variety of plants, organelles concluded that both cytoskeletal systems act indepen- migrate to the equatorial region after the first meiotic dently during metaphase-telophase. Our results, how- division, thus marking the future division site (Brown & ever, are supported by a number of recent reports on Lemmon, 1987, and references therein). The cytoplas- mitotic plant cells showing that actin does associate with mic disk of F-actin could obviously function in such a microtubules throughout division (Traas et al. 1987; process, re-distributing the cytoplasm in preparation for Schmit et al. 1985; Kakimoto & Shibaoka, 1987) and the cytokinesis. (2) The equatorial F-actin also extends to the conflicting evidence could simply reflect differences in plasma membrane where the network seems to be more stability of the mitotic actin system under different dense. The disk of actin is therefore continuous across the preparative conditions. cell from one side to the other. As such it would It has been proposed that actin could function in inevitably 'memorize' the division plane at the cortex. A chromatid separation (Forer et al. 1979; Cande et al. similar role has been proposed for the microtubular 1977; Seagull et al. 1987). However, this is not firmly preprophase band (PPB), which accurately marks the established and it appears from in vitro experiments that future site of division at the cortex of polarized cells microtubule depolymerization is sufficient to explain (Lloyd, 1987; Traas, 1988, for reviews). At one time it chromosome movement without the need for actin as a was thought that microtubules within the PPB were alone force generator (e.g. see Koshland et al. 1988; Gorbsky et responsible for marking the division site. Now it is known al. 1988). Our results indicate a completely different role that F-actin also occurs within that band (Palevitz, 1987; for actin in mitosis: cytochalasins perturb spindle forma- Traas et al. 1987) as well as in the plane of the future tion and therefore actin could be involved in spindle division site (Lloyd & Traas, 1988). It is important to formation or organization, i.e. in the reorganization of appreciate that there is no PPB of microtubules in the microtubular array during division. Likewise, meiocytes to mark the division site. Perhaps the actual Kobayashi et al. (1987) have proposed that actin fila- plane of division in meiocytes is not initially fixed as it is ments are actively involved in the re-alignment of micro- in somatic cells. However, it is clear that the actin tubules during differentiation of tracheary elements of network alone is sufficient to form a raft that bi-lateralizes Zinnia. In these cells microtubules and actin filaments the daughter nuclei and delineates the first division plane, are co-parallel. The microtubules switch their orientation which is to be followed by another at right angles. The from longitudinal to transverse during re-differentiation, absence of a PPB accentuates the involvement of F-actin but cytochalasin B inhibits microtubular re-orientation. in these division processes. In cytokinesis in animal cells

548 jf. A. Traas et al. F-actin is concentrated in the cortical division site (the important in the next phase in which the molecular basis contractile ring) before and during furrowing. It has been of meiosis will be studied. argued that astral arrays of microtubules radiating out to the equatorial region determine the division planes (re- References view: Mabuchi, 1986). In plant meiocytes the microtubules radiating out from the daughter nuclei to the BROWN, R. & LEMMON, B. E. (1987). Division polarity, development future division site could function in a similar way, and configuration of microtubule arrays in Bryophyte meiosis. perhaps by transporting or guiding the actin network to Protoplasma 138, 1-10. the equator. CANDE, W. Z., LAZARIDES, E. & MCINTOSH, J. R. (1977). A comparison of the distribution of actin and tubulin in the mammalian mitotic spindle as seen by indirect The second meiotic division: spindle and phragmoplast immunofluorescence. J. Cell Biol. 72, 552-567. formation CHAMBONNET, D. & DUMAS DE VAULX, R. (1983). A new anther culture medium performant on various eggplant (Solatium During prophase II, microtubules extending freely into melongena L.) genotypes. Proc. Vth Meet, of the Capsicum and the cytoplasm between the daughter nuclei start to Eggplant Working Group, 4-7 July, 1983 (ed. Daskalov), depolymerize, whereas those between the nuclear envel- pp. 38-41. Bulgaria: Plovdiv. ope and the plasma membrane elongate. As in prophase CLAYTON, L. & LLOYD, C. W. (1984). The relationship between the division plane and spindle geometry in Allium cells treated with I, interactions with the membrane could help to stabilize CIPC and griseofulvin: an anti-tubulin study. Eur.J. Cell Biol. 34, certain classes of microtubules, which then could partici- 248-253. pate in spindle formation/alignment. DE BRABANDER, M., GUEUENS, G., NUYDENS, R., WILLEBRORDS, After nuclear division, the microtubules again radiate R., AERTS, F. & DE MEY, J. (1986). Microtubule dynamics during the cell cycle: the effects of taxol and nocodazole on the out from the nuclear envelope in telophase II. F-actin microtubule system of PtK2 cells at different stages of the mitotic invades the future division planes as it did after the first cycle. Int. Rev. Cytol. 101, 215-274. division, but this time it remains there while the phrag- DICKINSON, H. G. (1988). The physiology and biochemistry of moplast develops. Comparing the two meiotic divisions, meiosis in the anther. Int. Rev. Cytol. 107, 79-108. FORER, A., JACKSON, W. T. & ENGBERG, A. (1979). Actin in spindles it seems that the concentration of actin in the division of Haemanthus katherinae endosperm II. Distribution of actin in planes has two distinct roles. First, the F-actin disk could chromosomal spindle fibers, determined by analysis of serial be involved in re-distributing the cytoplasm in prep- sections. J. Cell Sci. 37, 349-371. aration for cytokinesis as was suggested above. Such a GORBSKY, G. J., SAMMAK, P. J. & BORISY, G. G. (1988). process would be independent of the formation of a cross- Microtubule dynamics and chromosome motion visualized in living anaphase cells. J. Cell Biol. 106, 1185-1192. wall and constitutes a distinct step during meiosis. GUNNING, B. E. S. & HARDHAM, A. R. (1982). Microtubules. A. During the second division, F-actin could initially func- Rev. PI. Physiol. 33, 651-698. tion in the same way but, in remaining, would have the HOGAN, C. (1987). Microtubule patterns during meiosis in two additional role of guiding the outgrowth of the phragmo- higher plant species. Protoplasma 138, 126-136. HUSSEY, P. J., TRAAS, J. A., GULL, K. & LLOYD, C. W. (1987). plast as proposed for other plant cells (Schmit & Lam- Isolation of cytoskeletons from synchronized plant cells: the bert, 1987; Lloyd & Traas, 1988). This suggestion is also interphase array utilizes multiple tubulin isotypes. J. Cell Sci. 88, supported by the fact that cytochalasin treatments per- 225-230. turb the alignment of division planes and formation of the HYMAN, A. A. & WHITE, J. G. (1987). Determination of cell division phragmoplast. It is an interesting aspect of meiosis that axes in the early embryogenesis of Caenorhabditis elegans. J. Cell Biol. 105, 2123-2135. apparently the cell is able to uncouple these otherwise JOHN, B. & LEWIS, K. R. (1965). The Meiotic System, closely linked steps, thus preventing phragmoplast for- Protoplastomologica, vol. 6. Wien, New York: Springer Verlag. mation. KAKIMOTO, T. & SHIBAOKA, H. (1987). Actin filaments and microtubules in preprophase band and phragmoplast of tobacco In summary, our results suggest that the microtubular cells. Protoplasma 140, 151-156. system primarily acts in the establishment of spindle and KILMARTIN, J. V., WRIGHT, B. & MILSTEIN, C. (1981). Rat cell polarity and that F-actin is involved in the establish- monoclonal antitubulin antibodies derived by using a new non- secreting rat cell line. J. Cell Biol. 93, 576-582. ment and 'memorization' of the division planes and in the KOBAYASHI, H., FUKUDA, H. & SHIBAOKA, H. (1988). Interrelation organization of the cytoplasm during meiosis. Moreover, between the spacial disposition of actin filaments and microtubules reorganization of the cytoskeleton greatly depends on the during the differentiation of tracheary elements in cultured Zinnia interaction between the two systems. These seem to be cells. Protoplasma 140, 151-156. KOSHLAND, D. E., MlTCHlSON, T. J. & KlRSCHNER, M. W. (1988). general features not only of plant cell division but, as Polewards chromosome movement driven by microtubule discussed above, also of animal cell division. depolymerization in vitro. Nature, Loud. 331, 499-504. It is tempting to describe processes like cell division LLOYD, C. W. (1987). The plant cytoskeleton: the impact of entirely in terms of microtubule dynamics. Yet, a number fluorescence microscopy. A. Rev. PI. Physiol. 38, 119-139. of observations including our own also stress the import- LLOYD, C. W. (1988). Actin in plants. J. Cell Sci. 90, 185-188. LLOYD, C. W. & TRAAS, J. A. (1988). The role of F-actin in ance of microtubule—actin interactions in plant cell determining the division plane of carrot suspension cells. Drug division and it is likely that the cytoskeleton as a whole is studies. Development 102, 211-221. involved in the spatial control of cell division. MABUCHI, I. (1986). Biochemical aspects of cytokinesis. Int. Rev. Meiosis offers an important tool for analysing the Cytol. 101, 175-213. OLIVER, J. M., KRAWIEC, J. A. & BERLIN, R. D. (1978). A coordination of cell division. Populations of meiocytes carbamate herbicide causes microtubule and microfilament can be obtained in which every cell is in division. Because disruption and nuclear fragmentation in fibroblasts. Expl Cell Res. of their synchronous development, such cells will be 116, 229-237.

Eggplant cytoskeleton during meiosis 549 PALEVTTZ, B. A. (1980). Comparative effects of phalloidin and male meiosis in Lilium. Cell Biol. Int. Rep. 12, 471-476. cytochalasin B on motility and morphogenesis in Allimii. Can. J. SYBENGA, J. (1975). Meiotic Configurations. Berlin, Heidelberg, New Bot. 58, 773-785. York: Springer Verlag. PALEVTTZ, B. A. (1987). Actin in the preprophase band of Allium TRAAS, J. A. (1989). The plasma membrane associated cytoskeleton. cepa.J. CellBiol. 104, 1515-1519. In The Plant Plasma Membrane: Structure, Function and SCHMIT, A. C, VANTARD, M. & LAMBERT, A. M. (1985). Molecular Biology (ed. C. Larsson & I. M. Moller). (in press). Microtubule and F-actin rearrangement during the initiation of TRAAS, J. A., DOONAN, J. H., RAWLINS, D. J., SHAW, P. J., mitosis in acentriolar higher plant cells. In Cell Motility: WATTS, J. & LLOYD, C. W. (1987). An actin network is present in Mechanisms and Regulation (ed. H. Ishikawa, S. Hatano & H. the cytoplasm throughout the cell cycle of carrot cells and Sato), pp. 415-433. Tokyo: University Press. associates with the dividing nucleus. J. Cell Biol. 105, 387-395. SCHMIT, A. C. & LAMBERT, A. M. (1987). Characterization and VAN LAMMEREN, A. A. M., KEIJZER, C. J., WILLEMSE, M. T. M. & dynamics of cytoplasmic F-actin in higher plant endosperm cells KJEFT, H. (1985). Structure and function of the microtubular during interphase, mitosis, and cytokinesis, J. Cell Biol. 105, cytoskeleton during pollen development in Gastena verucosa 2157-2166. (Mill.) H. Duval. Planta 165, 1-11. SEAGULL, R. W., FALCONER, M. M. & WEERDENBURG, C. (1987). WEERDENBURG, C, FALCONER, M. M., SETTERFIELD, G. & SEAGULL, Microfilaments: dynamic arrays in higher plant cells. J. Cell Biol. R. W. (1986). Effects of taxol on microtubule arrays in cultured 104, 995-1004. higher plant cells. Cell Motil. Cytoskel. 6, 469-478. SHELDON, J. M. & DICKINSON, H. G. (1986). Pollen wall formation in Ulium. The effect of chaotropic agents and the organisation of the microtubular cytoskeleton during pattern development. Planta 158, 11-23. (Received J September I9SS - Accepted, in revised form, SHELDON, J. M. & HAWES, C. (1988). The actin cytoskeleton during 4 January 1989)

550 J. A. Traas et al.