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 div- ision, 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.