the role of anillin in cytokinesis - journal of cell sciencering to the cell cortex. moreover, the...

INTRODUCTION

In animal cells cytokinesis is accomplished by the contractionof a ring-shaped cellular structure containing actin and myosinII filaments. This structure is anchored to the plasmamembrane at the equator of the dividing cell and constricts itlike a purse string, leading to the separation of the two daughtercells (reviewed by Fishkind and Wang, 1995; Glotzer, 1997;Goldberg et al., 1998). Although this ‘contractile ring’ modelis the commonly accepted mechanism for cytokinesis, there areseveral questions that remain to be answered. For example,little is known about the structural and regulatory proteins thatcompose the contractile ring in addition to actin and myosinII, and about the molecules that mediate the anchoring of thering to the cell cortex. Moreover, the molecular mechanismsunderlying the assembly, contraction and disassembly ofcontractile ring are still poorly understood. Finally, althoughthere is evidence that the spindle dictates the positioning andtiming of ring assembly, the molecules involved in thesesignaling pathways have not been identified.

Drosophila melanogaster male meiosis provides anexcellent model system for the genetic, molecular andcytological analysis of cytokinesis (Cenci et al., 1994;Williams et al., 1995; Gunsalus et al., 1995; Giansanti et al.,1996; Hime et al., 1996; Basu et al., 1998; Carmena et al.,1998; Giansanti et al., 1998; Herrmann et al., 1998). In

Drosophila males four gonial divisions generate 16 primaryspermatocytes which remain connected by cytoplasmic bridgescalled the ring canals (henceforth abbreviated as RCs). Thesecells, engulfed in a cyst formed by two specialized cyst cells,undergo a dramatic growth phase that results in a 25-foldincrease in nuclear volume. Mature spermatocytes entermeiosis synchronously and produce 64 spermatids which alsoremain connected by RCs (Lindsley and Tokuyasu, 1980;Fuller, 1993; Hime et al., 1996). Each spermatid consists of anucleus and a mitochondrial derivative called the nebenkern.During both meiotic divisions mitochondria associatelengthwise along the central spindle and are equally partitionedbetween the two daughter cells when cytokinesis occurs. Thus,newly formed ‘onion stage’ spermatids have sphericalnebenkerns of constant dimensions, each associated with anucleus of similar shape and size (Lindsley and Tokuyasu,1980; Fuller, 1993; Cenci et al., 1994). Failures of meioticcytokinesis can be easily detected because they result inspermatids containing abnormally large nebenkerns associatedwith two or four normally-sized nuclei (Fuller, 1993; Castrillonand Wasserman, 1994; Williams et al., 1995; Gunsalus et al.,1995; Carmena et al., 1998; Giansanti et al., 1998).

Examination of male meiotic and postmeiotic stages notonly provides a way to assess failures in cytokinesis but mayalso reveal the primary defects that impair the cytokineticprocess. Due to the relatively large size of primary

2323Journal of Cell Science 112, 2323-2334 (1999)Printed in Great Britain © The Company of Biologists Limited 1999JCS0470

Anillin is a 190 kDa actin-binding protein that concentratesin the leading edges of furrow canals during Drosophilacellularization and in the cleavage furrow of both somaticand meiotic cells. We analyzed anillin behavior during D.melanogaster spermatogenesis, and focused on therelationships between this protein and the F-actin enrichedstructures. In meiotic anaphases anillin concentrates in anarrow band around the cell equator. Cytological analysisof wild-type meiosis and examination of mutants defectivein contractile ring assembly (chickadee and KLP3A),revealed that the formation of the anillin cortical bandoccurs before, and does not require the assembly of the F-actin based contractile ring. However, once the acto-myosinring is assembled, the anillin band precisely colocalizeswith this cytokinetic structure, accompanying itscontraction throughout anaphase and telophase. In

chickadee and KLP3A mutant ana-telophases the corticalanillin band fails to constrict, indicating that its contractionis normally driven by the cytokinetic ring. These findings,coupled with the analysis of anillin behavior in twinstarmutants, suggested a model on the role of anillin duringcytokinesis. During anaphase anillin would concentrate inthe cleavage furrow before the assembly of the contractilering, binding the equatorial cortex, perhaps through itscarboxy-terminal pleckstrin homology (PH) domain.Anillin would then interact with the actin filaments of theacto-myosin ring through its actin-binding domain,anchoring the contractile ring to the plasma membranethroughout cytokinesis.

Key words: Anillin, Cytokinesis, Contractile ring, Ring canal,Fusome, Drosophila, Spermatogenesis

SUMMARY

The role of anillin in meiotic cytokinesis of Drosophila males

Maria Grazia Giansanti1, Silvia Bonaccorsi1,2 and Maurizio Gatti1,2,*1Istituto Pasteur-Fondazione Cenci Bolognetti and 2Centro di Genetica Evoluzionistica del CNR, Dipartimento di Genetica eBiologia Molecolare, Universita’ di Roma ‘La Sapienza’, P.le A. Moro 5, 00185 Roma, Italy*Author for correspondence

Accepted 11 May; published on WWW 24 June 1999

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spermatocytes, meiotic spindles are very prominent and can bereadily detected by tubulin immunostaining. In wild-typemales both meiotic divisions exhibit conspicuous centralspindles which are pinched in the middle during telophase(Cenci et al., 1994). These cells also exhibit an actin-basedcontractile ring around the central spindle midzone wherepinching occurs (Gunsalus et al., 1995; Hime et al., 1996;Giansanti et al., 1998).

Several mutations have been identified which disruptmeiotic cytokinesis and affect specific cellular structuresinvolved in this process. For example, mutations in KLP3A, agene encoding a kinesin-like protein that accumulates at thecentral spindle midzone, disrupt the central spindle formationand suppress contractile ring assembly (Williams et al., 1995;Giansanti et al., 1996, 1998). Similar meiotic phenotypes areproduced by mutations in chickadee (chic) and diaphanous(dia) (Giansanti et al., 1996, 1998). chic encodes a Drosophilaprofilin, a low molecular mass protein that regulates actinpolymerization (Cooley et al., 1992); the Dia polypeptide ishomologous to the limb deformity gene product of mouse, tothe BNI1 protein of Saccharomyces cerevisiae and to the Cdc12 protein of Schizosaccharomyces pombe (Castrillon andWasserman, 1994; Chang et al., 1996). The Cdc 12 proteininteracts with profilin through its prolin-rich domain and isrequired for both actin ring formation and cytokinesis (Changet al., 1997). These findings have suggested a cooperativeinteraction between the central spindle microtubules andelements of the cortical actin cytoskeleton involved in theassembly of the contractile ring (Giansanti et al., 1996, 1998).

A simultaneous absence of the central spindle and thecontractile ring, accompanied by a failure in cytokinesis, hasbeen also observed in embryonic cells of mutants in thepavarotti (pav) locus. pav encodes a kinesin-like protein (PAV-KLP) which forms a complex with the POLO kinase andconcentrates in the central spindle midzone during telophase(Adams et al., 1998). These findings indicate that theinterdependence of the central spindle and the contractile ringmay be a common feature of both meiotic and somatic cells.Interestingly, meiotic cells of polo males also exhibit defectsin both the central spindle and the contractile ring, resulting infrequent disruptions of cytokinesis (Carmena et al., 1998).Because the PAV-KLP and POLO proteins appear to bemutually dependent for their correct localization, thecytological phenotype observed in polo meiosis may be aconsequence of the incorrect localization of either protein(Carmena et al., 1998).

While KLP3A, chic, dia and polo are required for contractilering assembly, twinstar (tsr) is primarily involved in thedisassembly of this structure (Gunsalus et al., 1995). tsrencodes a polypeptide homologous to the cofilins, a class ofproteins that can sever and depolymerize actin filaments invitro (reviewed by Moon and Drubin, 1995). In tsr mutantscontractile rings are initially morphologically regular andundergo a normal contraction. However, at the end of eachmeiotic division, they become abnormally shaped, fail todisassemble and form large and persistent F actin aggregateswhich interfere with the proper execution of cytokinesis(Gunsalus et al., 1995).

Another protein that is likely to play an important role inmeiotic cytokinesis of Drosophila males is anillin. Anillin is a190 kDa protein isolated by actin filament cromatography of

Drosophila embryo extracts (Miller et al., 1989). Molecularanalysis of the anillin gene showed that its product is nothomologous to known polypeptides. However, in vitro studiesrevealed that anillin can bundle actin filaments through anovel actin-binding domain. Indirect immunofluorescenceexperiments using antibodies to anillin showed that this proteinconcentrates in the leading edges of furrow canals duringcellularization and in the cleavage furrows of a variety ofDrosophila cell types, including tissue culture cells, gonialcells of both sexes and male meiotic cells (Field and Alberts,1995; Hime et al., 1996; de Cuevas and Spradling, 1998). Inaddition, anillin accumulates in the walls of RCsinterconnecting developing germ line cells of both sexes, andin somatic RCs that link ovarian follicle cells (Field andAlberts, 1995; Hime et al., 1996; de Cuevas and Spradling,1998).

In the present paper we have analyzed anillin behaviorduring Drosophila spermatogenesis, with a particular focus onits role during meiotic cytokinesis. Moreover, we haveexamined by immunofluorescence the relationships betweenanillin and actin in mutants with altered actin behavior, suchas KLP3A, chic and tsr. The results of our analyses indicatethat anillin concentrates in the cleavage furrow during gonialmitoses and meiotic divisions, and in the RCs that interconnectgerm cells. Anillin is not always associated with F-actin butexhibits a dynamic pattern of F-actin binding. Our data areconsistent with the hypothesis that anillin concentrates inspecialized cortical sites with an unknown mechanism,mediating the anchoring of F-actin to these sites at particulartimes of the cell cycle. Our studies on the relationships betweenanillin and F-actin have also provided detailed information onthe organization and development of the male fusome.

MATERIALS AND METHODS

Drosophila stocksImmunocytological analyses of wild-type spermatogenesis were madeusing an Oregon-R stock that has been maintained in our laboratoryfor about 30 years. Observations on KLP3A mutants were performedusing the KLP3Ae4 allele which carries a deficiency that removes 82amino acids at the COOH terminus of the KLP3A protein (Williamset al., 1995). To obtain KLP3Ae4 males, KLP3Ae4/FM7 virgin femaleswere mated with FM7 males and their progeny scored for Malpighiantubule coloration (Williams et al., 1995).

Analyses on chic and tsr were performed using the chic R1, chic35A

(Giansanti et al., 1998) and tsr1 mutant alleles (Gunsalus et al., 1995).These mutations were all induced by single P element mutagenesisand were kept over the compound balancer ST, kindly provided by DrGarcia-Bellido, Madrid. ST is a translocation between the secondchromosome balancer SM6a and the third chromosome balancerTM6b, that carries the body-shape marker Tubby (Tb) and thedominant marker Curly (Cy). Homozygous mutant larvae and pupaewere distinguished from their heterozygous sibs for their non-Tubbyphenotype.

The flies were reared on standard Drosophila medium at 25+1°C.

Fixation proceduresDissection, fixation and staining, if not otherwise specified, wereperformed at room temperature. Cytological preparations were madewith testes from third instar larvae or young pupae, using threedifferent procedures. In two of these procedures, testes were dissectedin testis isolation buffer (183 mM KCl, 47 mM NaCl, 10 mM Tris-

M. G. Giansanti, S. Bonaccorsi and M. Gatti

2325The role of anillin in cytokinesis

HCl, pH 6.8), gently squashed in 2 µl of the same buffer under a20×20 coverslip, and frozen in liquid nitrogen. After the removal ofthe coverslip, testis preparations were fixed according to either of thefollowing protocols: (1) For simultaneous immunostaining with anti-α-tubulin and anti-anillin antibodies, the slides were fixed by coldmethanol and acetone according to the method of Cenci et al. (1994).(2) For F-actin staining with phalloidin the slides were fixed with3.7% formaldeyde according to the method of Gunsalus et al. (1995).For simultaneous immunostaining with anti-α-tubulin and anti-anillinantibodies, we also used the following procedure (protocol 3). Testeswere dissected in saline (0.7% NaCl in distilled water) and fixed in

3.7% formaldehyde in PBS for 30 minutes. They were rinsed for 30seconds in 45% acetic acid and transferred into a drop (4 µl) of 60%acetic acid placed on a 20×20 coverslip. Testes were kept in 60%acetic acid for 2-3 minutes and then squashed applying a moderatepressure. Slides were frozen in liquid nitrogen and, after removal ofthe coverslip, immersed in ethanol at −20°C for 15 minutes. Slideswere then incubated in PBT (PBS containing 0.1% Triton-X) for 10minutes, washed 2 times (5 minutes each) in PBS and air dried.

Staining proceduresDouble and triple stainings were always performed in the following

Fig. 1. Fusome development during Drosophila spermatogenesis. The merged images were obtained by overlapping the anillin and F-actinsignals. (A) A 4-cell gonial cyst with 3 RCs traversed by the fusome. The anillin not associated with RCs is concentrated in the nucleus butexcluded from the area occupied by the chromatin. Note that the cyst cell nuclei (arrows) contain less anillin than the spermatogonial nuclei.(B) An 8-cell gonial cyst with a growing fusome. The fusome elements associated with the newly formed RCs are not yet connected with thecentral fusome. Anillin has just begun to enter the nuclei and forms a thin layer around the chromatin. The arrow points to the two cyst cellswhich are very close to each other. (C) A cyst containing 16 young primary spermatocytes connected by 15 RCs. Seven of these RCs aretraversed by a preexisting fusome, while the 8 newly formed RCs exhibit actin-enriched, growing fusome elements. The anillin not associatedwith RCs has a cytoplasmic localization. The cyst cells cannot be unambiguously recognized. (D) A partial cyst containing 4 primaryspermatocytes in the S3 stage and 3 ring canals connected by the fusome. Anillin is concentrated in the nucleus and excluded from thenucleolus. Bar, 5 µm.

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order: immunostaining, phalloidin staining and Hoechst 33258staining. Before immunostaining, fixed preparations were incubatedwith 1% BSA (Sigma) in PBS for 45 minutes. All subsequentincubations with antibodies were performed in a humid chamber inthe dark.

For anillin localization two different rabbit antisera were used asprimary antibodies, one raised against amino acids 1-371 and the otheragainst amino acids 401-828 (Field and Alberts, 1995), both diluted1:300 in 1% BSA in PBT. After overnight incubation at 4°C, the slideswere washed twice in PBT and once in PBS, for a total of 15 minutes.The primary antibodies were then detected by incubation for onehour with either tetramethylrhodamine isothiocyanate- (TRITC)conjugated anti-rabbit IgG (Cappel), diluted 1:50 in PBT, or withfluorescein isothiocyanate- (FITC) conjugated anti-rabbit IgG(Cappel), diluted 1:50 in PBT. Regardless of the protocol used forfixation, these two antisera produced identical immunostainingpatterns for all cell types in Drosophila spermatogenesis.

For simultaneous visualization of anillin and microtubules, testispreparations were first immunostained with anti-anillin antibodies asdescribed above; they were then treated for 45 minutes with amonoclonal anti-α-tubulin antibody (Amersham; Blose et al., 1982),diluted 1:50 in PBS. After two washes in PBS (5 minutes each), theslides were incubated with the secondary antibody (sheep anti-mouseIgG F(ab′)2 fragment conjugated with 5(6)-carboxy-fluorescein-N-hydroxysuccinimide ester (FLUOS), Boehringer), diluted 1:10 inPBS.

For actin staining, testis preparations were incubated withrhodamine-labelled phalloidin (Molecular Probes) dissolved in PBS(100 units/µl) for 1 hour and 30 minutes at 37°C in the dark, and thenrinsed for 2 minutes in PBS. To prepare the phalloidin solution, analiquot of a stock solution of rhodamine-labelled phalloidin inmethanol (300 units in 1.5 ml of methanol) was vacuum dried andresuspended in the appropriate volume of PBS.

After immunostaining, or immunostaining plus actin staining, testispreparations were air dried and stained with Hoechst 33258 accordingto the method of Bonaccorsi et al. (1988).

Microscopy and image analysisAll preparations were examined with a Zeiss Axioplan microscopeequipped with an HBO 50W mercury lamp (Osram) forepifluorescence, and with a cooled charge-coupled device (CCD;Photometrics). Hoechst 33258, FLUOS or FITC, and TRITCfluorescence were detected using the 0.1 (BP 365/11, FT 395, LP397), 10 (BP 450/490, FT 510, LP 515/565) and 15 (BP 546, FT 580,LP 590) Zeiss filter sets, respectively. Gray-scale digital images werecollected separately using the IP Lab Spectrum software, converted toPhotoshop 2.5 format (Adobe), pseudocoloured and merged. We alsorecorded the phase-contrast images underlying the various types ofimmunostained preparations. The combined examination of theseimages with those produced by fluorescent stainings enabled us tounambiguously recognize the various stages into which Drosophilaspermatogenesis has been subdivided (Cenci et al., 1994).

RESULTS

Anillin localization in premeiotic stagesTo analyze anillin localization in spermatogonia we examinedcysts containing 2, 4 or 8 cells. The Hoechst 33258 stainingpattern (Cenci et al., 1994) and the developmental stage ofthe fusome (see below) permitted unambiguous distinctionbetween cysts containing cells in early interphase and thosewith late interphase cells. In late interphase spermatogoniaanillin is localized primarily within the nucleus but it isexcluded from the chromatin. Because in these cells

chromatin occupies most of the nuclear space, anillin isusually concentrated in a narrow layer at the periphery of thenucleus (Fig. 1A). During gonial divisions anillin is releasedin the cytoplasm and concentrates in the cleavage furrow atanaphase and telophase (not shown). At the end of eachgonial division the two daughter cells remain connected by aRC which is a derivative of the contractile ring (Hime et al.,1996). A fraction of the anillin accumulated in the cleavagefurrow remains in the RC, while another, larger fraction ofthis protein becomes diffuse in the cytoplasm. Thus, inrecently divided, early interphase gonial cells anillin has apredominant cytoplasmic localization (not shown). However,as the cell cycle proceeds, it progressively migrates into thenucleus (Fig. 1B).

Anillin also has a cytoplasmic localization in very youngspermatocytes (Fig. 1C) but, here again, it rapidly concentratesinto the nucleus as these cells enter their growth phase. Duringspermatocyte growth anillin remains confined to the nucleusand to the RCs (Figs 1D and 2). In early stages of spermatocytegrowth (stages S1-S4; see Cenci et al., 1994, for stagenomenclature) anillin tends to be excluded from the nucleolus(Fig. 2). However, in mature spermatocytes (stage S5) anillinprogressively concentrates in this structure, but is releasedagain into the nucleoplasm when the nucleolus breaks downduring the S6 stage (Figs 2 and 3).

Previous studies have shown that anillin consistentlydecorates the male RCs. These structures develop from arrestedcontractile rings after a specialized type of cytokinesis in whichthe closing of the invaginating plasma membrane is incomplete(Hime et al., 1996). In each cyst the number of RCs is thereforeequal to the number of germ line cells contained in the cystminus one. Thus, gonial cysts of 2, 4, and 8 cells and primaryspermatocyte cysts of 16 cells contain 1, 3, 7 and 15 RCs,respectively (Hime et al., 1996; Figs 1 and 2). Interestingly,once formed, these RCs persist without substantialmorphological changes throughout spermatogenesis (Hime etal., 1996; see below).

F-actin staining by phalloidin and anillin immunostainingrevealed that RCs are often traversed by a ribbon-like structurecontaining F-actin. This structure, which has been alreadyidentified as the male fusome (Gunsalus et al., 1995; Hime etal., 1996), exhibits a characteristic developmental pattern (Fig.1). In 2-, 4- and 8-cell cysts, when spermatogonia are about toenter mitotic division, all the RCs present in any given cyst aretraversed by the fusome. At the end of cell division, the newlyformed RCs are very similar but not identical to contractilerings (see also below). Whereas in contractile rings F-actin andanillin colocalize precisely, in new RCs F-actin tends to beslightly dislocated with respect to anillin. In particular, in somecases we can see small actin-rich masses protruding from oneor both sides of the anillin rings. As cells proceed through thecell cycle, these actin ‘plugs’ progressively extend, andeventually join to the preexisting fusome. For example, a 4-cellgonial cyst, before entering mitosis, contains 3 RCs connectedby the fusome (Fig. 1A). After completion of cell division, the8 early interphase cells contained in the cyst exhibit 3 RCstraversed by the fusome and 4 newly formed RCs (Fig. 1B).As cell cycle progresses these 4 RCs emanate new fusomeelements which eventually become connected to thepreexisting fusome.

The 16-cell primary spermatocyte cysts exhibit a similar



pattern of fusome development. In very young spermatocytes(stages S0 and S1) the fusome connects 7 RCs, while the 8new RCs are not associated with this structure (Fig. 1C). Hereagain, as spermatocyte growth proceeds, these 8 RCs becometraversed by newly formed actin-rich fusome elements whichprogressively conjoin to the preexisting ones, forming acontinuous branched structure. By the S3 stage, in most cystsall the 15 RCs are connected by the fusome (Fig. 1D). Theprimary spermatocyte fusome persists till the S4 stage andthen gradually disassembles. In mature spermatocytes at theS5 stage the fusome regresses and breaks down into pieces(Fig. 4).

Anillin behavior in meiotic and postmeiotic stagesIn the M1a and M1b stages, when chromatin has alreadyattained a high degree of condensation and two prominentasters are present, anillin is still concentrated in the nucleus(Fig. 3). In the M2 prometaphase stage, when the lamin-based nuclear envelope dissolves (Cenci et al., 1994; White-Cooper et al., 1993; Eberhart and Wasserman, 1995), anillindiffuses in the cytoplasm (Fig. 3). During metaphase (stageM3) and early anaphase I (stage M4a) anillin remains diffusewithin the cell (Fig. 5A,D). However, at mid-anaphase (stageM4b) anillin concentrates in a narrow circumferential bandaround the equator of the cell (Figs 5B,E, 6A). Simultaneousstaining with phalloidin and anti-anillin antibodies revealedthat the anillin band appears earlier than the actin ring (Fig.6). Anillin accumulation at the cleavage furrow is clearlyvisible when the cell is in mid-anaphase and the centralspindle has just begun to assemble (stage M4b; Figs 5B,E,6A). The F-actin ring becomes first detectable when thedividing cell is further elongated and the central spindleis fully formed (Fig. 6B). Remarkably, since its firstappearance, the actin ring colocalizes with the anillincortical band. These two structures remain strictly associatedtill the completion of acto-myosin ring contraction (stageM5; Figs 5C,F, 6C).

Interestingly, in preparations fixed after testis squashing andfreezing (protocols 1 and 2; see Materials and Methods), theanillin band encircling mid-anaphase cells (stage M4b) is ofteninterrupted and/or dislocated from the cell equator (Figs 5B,E,6A). However, if preparations are fixed before squashing andfreezing (protocol 3; see Materials and Methods), the anillinband of M4b cells is usually continuous and consistentlylocated at the cell equator (Fig. 7A,B). Regardless of the typeof fixation, when anillin becomes associated with the actin ringit is almost invariably organized in a continuous structurewhich encircles the central spindle midzone (Fig. 6B). Themost obvious interpretation of these findings is that thediscontinuity and mislocation of the anillin band observed inM4b cells fixed according to either protocol 1 or 2, is due tothe rupture of the plasma membrane with which anillin isassociated, during the squashing/freezing procedure. M4b cellstreated with formaldheyde before squashing (protocol 3) arehardened by the fixation, and are thus likely to be much lesssubject to rupturing than cells squashed and frozen beforefixation. Similarly, when the anillin band is tightly associatedwith the acto-myosin ring, it is likely to be resistant to damagecaused by squashing and freezing.

At the end of the first meiotic division the contractile ringreorganizes into a RC. Here again, only a fraction of the anillin

initially accumulated in the cleavage furrow remains in the RC(Fig. 5). Most of the anillin associated with the cytokineticapparatus, during the isovolumetric contraction of theactomyosin ring, diffuses in the cytoplasm.

During telophase I the two daughter nuclei reform a nuclearenvelope, becoming sharply demarcated from the cytoplasm.This nuclear/cytoplasmic demarcation persists through theshort interphase between the first and the second meioticdivision (stage M6) and early prometaphase II (stage M7), butit disappears when cells enter late prometaphase II (stage M8).Despite the presence of a regular nuclear envelope, telophaseI cells and secondary spermatocytes at the M6-M7 stages donot accumulate anillin into the nucleus; in these cells the anillinnot associated with RCs is dispersed in the cytoplasm (Fig.5G). In meiotic prometaphase, metaphase and early anaphaseII (stages M8-M10a) anillin remains dispersed in thecytoplasm; it concentrates in a cortical band at the equator ofthe cell during mid-anaphase (stage M10b). Here again, theanillin band appears earlier than the actin ring which firstbecomes detectable in stage M10c. In subsequent meioticstages anillin behaves just as in the first meiotic division andeventually becomes a component of the newly formed RCs(Fig. 5).

In postmeiotic stages the overall amount of anillin detectableby immunofluorescence appears to be substantially reducedwith respect to the previous stages of germ cell differentiation.Little or no anillin appears to be present either in the cytoplasmor in the spermatid nucleus (Fig. 8). However, anillin remainsassociated with the spermatid RCs which appear to contain thesame amount of anillin as those of premeiotic and meioticstages (Fig. 8).

At the end of the second meiotic division spermatid cystscontain 63 RCs. While a few of these RCs are associated withpersisting fragments of the spermatocyte fusome, most of themhave the typical appearance of newly formed RCs (i.e. theycontain a small plug of actin enriched material, as shownin Fig. 1). As spermatogenesis proceeds, the spermatid RCsbecome progressively traversed by actin-rich ribbon-likestructures that eventually merge in a highly branched fusome(not shown). By the onion stage (T4 according to Cenci et al.,1994) most, if not all, the spermatid RCs are connected by thefusome.

Anillin behavior in mutants affecting cytokinesisTo obtain further insight into the interactions between anillinand F-actin during male meiosis, we immunostained with anti-anillin antibodies KLP3A, chic and tsr mutant testes. Asmentioned previously, mutations in KLP3A and chic suppressthe formation of both the central spindle and the contractilering (Williams et al., 1995; Giansanti et al., 1996, 1998). Inlate telophase, mutants in tsr exhibit morphologicallymisshaped contractile rings that fail to disassemble at the endof each meiotic division. These abnormal actin rings growslightly larger than the normal cytokinetic structures, andeventually become compact masses of F-actin (Gunsalus et al.,1995).

KLP3A and chic mutant testes exhibit similar anillinimmunostaining patterns. In both mutants anillin cycling fromthe nucleus to the cytoplasm is not altered. Moreover, duringthe M4b stage, anillin accumulates normally at the equatorof the dividing cells forming a circumferential band

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indistinguishable from that seen in wild type (Fig. 7). However,this anillin band fails to contract properly because most KLP3A(69/70) and chic (83/89) telophases fixed according to protocol3 (see Materials and Methods) exhibit an unconstricted anillinring. In telophases fixed with either protocol 1 or 2 this ring isoften discontinuous and/or displaced from the equator of thedividing cell (Fig. 8C). We have previously shown that about90% of KLP3A telophases and 95% of chicR1 telophasesfail to assemble an acto-myosin ring. Thus, the presentobservations indicate that the persistence of the anillin ringthroughout telophase does not require the presence of thecontractile ring. Yet, the acto-myosin ring appears to benecessary for proper contraction of the anillin band. Mostlikely, the discontinuity and the frequent displacement of theanillin ring in KLP3A and chic telophases fixed with eitherprotocol 1 or 2 are not direct consequences of these mutations.As mentioned above, we believe that these abnormalities areartifacts reflecting the lack of interaction between anillin andthe contractile ring. In the absence of an acto-myosin ring,anillin would be associated only with the plasma membrane,


Fig. 4. Fusome disintegration in a cyst containing mature primaryspermatocytes (S5 stage). (A) Hoechst 33258 staining (blue). (B) F-actin staining (red). Note in B the regression and fragmentation ofthe fusome. Bar, 5 µm.

Fig. 2. Anillin localization in primary spermatocytes. (A) A completecyst containing 16 primary spermatocytes in the S2 stage. Anillin isalready concentrated within the nuclei and excluded from thenucleolus; note the intense staining of the 15 RCs (arrows).(B) Partial cyst with two primary spermatocytes in the S5 stage; inthese cells most anillin has migrated into the nucleolus. Bar, 5 µm.

Fig. 3. Anillin localization during the M1-M2 stages of malemeiosis. (A,D) phase contrast; (B,E) microtubule immunostaining(green), and chromatin staining with Hoechst 33258 (blue);(C,F) anillin immunostaining (red). (A-C) Partial M1 cyst containingtwo nuclei, each surrounded by parafusorial and astral membranes(A), and associated with prominent meiotic asters (B). Note thatanillin is uniformly concentrated within the nuclei (C). (D-F) PartialM2 cyst. Nuclear-cytoplasmic demarcation has begun to disappear(D), some spindle fibres have penetrated the nucleus (E), and anillinis dispersed in the cytoplasm (F). Bar, 5 µm.


which would be subject to rupturing during the fixationprocedure.

During the second meiotic division of KLP3A and chicmutants anillin exhibits the same aberrant behavior displayedin the first meiotic division (not shown). As a consequence ofthe aberrant meiotic divisions, KLP3A and chic testes exhibitRCs of variable sizes. In both mutants the RCs present inspermatogonia and primary spermatocytes are usually normal.These apparently normal RCs persist through meiosis and arealso found in spermatids. However, spermatids also exhibitlarger, often severely misshaped RC-like structures (Fig. 8D).In chic and KLP3A testes the abnormally large RCs are usuallyassociated with spermatids composed by a large nebenkern and4 normally-sized nuclei, and represent 15% and 12% of thetotal RCs, respectively. This suggests that these large RCs are

Fig. 5. Anillin localization duringmeiotic divisions. Testispreparations were fixed accordingto protocol 1 (see Materials andMethods), and sequentially stainedwith anti-anillin and anti-α tubulinantibodies, and with Hoechst33258. (A-F) Anillin localizationduring the first meiotic division.(A-C) Anillin localization (red) inearly anaphase (A, stage M4a),mid-anaphase (B, stage M4b) andtelophase (C, stage M5). (D-F) Thesame anillin signals of A-C mergedwith tubulin (green) and Hoechst33258 (blue) stainings. In earlyanaphase (A,D) anillin is dispersedin the cytoplasm but concentrates inthe cleavage furrow from midanaphase (B,E) through telophase(C,F). (G-J) Merged images of thesecond meiotic division; colors asabove. In prometaphase (G, stageM8) and early anaphase (H, stageM10a) anillin is diffuse in thecytoplasm; note that anillin isexcluded from prometaphse nuclei.As in the first meiotic division, anillin concentrates in the cleavage furrow from mid anaphase (I, stage M10b) through telophase (J, stage M11).(A,D,G,H) Examples of RCs that persist through meiotic divisions. Bar, 5 µm.

Fig. 6. Anillin and actin localization during meiotic anaphase andtelophase I. Testis preparations were fixed according to protocol 2(see Materials and Methods) and sequentially stained with anti-anillin antibody (green), rhodamine-labelled phalloidin (red) andHoechst 33258 (blue). Note that Hoechst 33258 stains themitochondria associated with the central spindle, outlining thisstructure. During mid- anaphase (A, stage M4b) anillin is alreadyconcentrated in the equatorial cortex, while the F-actin ring is not yetvisible. From late anaphase (B, stage M4c) through telophase(C, stage M5) the F-actin and anillin signals precisely overlap. Bar,5 µm.

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derivatives of the abnormal anillin-enriched cytokineticstructures present in these mutants.

In KLP3A mutants both the structure and the dynamicbehavior of the fusome do not differ from wild type. We havepreviously described that in gonial and primary spermatocytecysts of chic mutants the fusome develops normally. However,it fails to disassemble properly during the S4 stage. As aconsequence, cysts of mature chic spermatocytes (stage S5)contain long-lived fusome remnants which disintegrate duringthe first meiotic division (Giansanti et al., 1998). Althoughspermatid cysts of chic mutants contain fewer RCs than wild-type cysts, they develop an apparently normal fusome thatconnects all the extant RCs.

In tsr mutant testes anillin cycling from the nucleus to thecytoplasm is completely normal, and anillin meiotic behaviordoes not substantially differ from that seen in wild type. Duringmid-anaphase of both meiotic divisions (stages M4b andM10b) of tsr mutants anillin forms a regular equatorial band.In late anaphase and telophase this band colocalizes with theF-actin ring and contracts along with it (Fig. 9). In latetelophase I and II, when the actin rings of tsr mutants fail todisassemble, overgrow and become misshaped, anillin nolonger colocalizes with F-actin but forms regularly-shapedrings which are often smaller than their wild-type counterparts(Figs 8E and 9B,C). The small size of these anillin rings islikely to be a consequence of the abnormal persistence of thecontractile ring in tsr mutants, which may result in a highercontraction of the anillin equatorial band.

An examination of tsr spermatid cysts revealed that theycontain two types of RCs: normally-sized RCs, and RCssmaller than their wild-type counterparts (23% of total RCs).Most of the RCs of normal size are probably formed duringthe gonial divisions. This is suggested by the observation thatthe RCs associated with tsr primary spermatocytes haveusually a normal size. The small RCs are likely to bederivatives of meiotic cleavage furrows which have attained ahigh degree of contraction due to the persistence of thecontractile apparatus.

We have already shown that in spermatogonial and primaryspermatocyte cysts of tsr mutants both the structure and thebehavior of the fusome are normal (Gunsalus et al., 1995).During the meiotic divisions of these mutants there is little orno fusome material, as occurs in wild type. In tsr spermatidcysts the fusome reforms but it is often interrupted and fails toconnect all the extant RCs (not shown). It is possible that thesedefects in fusome development are due to an insufficientintracellular concentration of actin. In tsr spermatid cysts asubstantial fraction of actin is trapped in the actin massesgenerated by the failure of disassembly of the meiosis IIcontractile rings (Gunsalus et al., 1995).

DISCUSSION

Anillin localization in interphase cellsField and Alberts (1995) have shown that anillin is present onlyin dividing cells, with the exception of ovarian nurse cellswhere anillin is presumably stored for later use duringembryogenesis. Accordingly, we have found that anillin isenriched in spermatogonia and spermatocytes but not in cellsthat have ceased dividing such as spermatids and sperm. Thus,

our findings reinforce the conclusion that the presence ofanillin in a cell correlates with its potential to divide (Field andAlberts, 1995).

In most types of somatic cells examined by Field andAlberts anillin concentrates in the nuclei in a cell cycle-dependent fashion; in recently divided cells anillin islocalized in the cytoplasm but it is imported into the nucleusas cells proceed through the cell cycle. Anillin alternatesbetween a nuclear and cytoplasmic localization also inspermatogonia and primary spermatocytes. In recentlydivided spermatogonia and very young primaryspermatocytes anillin is cytoplasmic but it becomes restrictedto the nucleus as these cells progress through the cell cycleor the growth phase. The mechanisms by which anillin isimported into the nucleus are currently unknown, althoughthey are likely to exploit the three potential nuclearlocalization sequences present in this protein (Field andAlberts, 1995). Also unknown is the biological meaning ofthe nuclear confinement of anillin. Another protein whichexhibits an intranuclear localization in spermatogonia andprimary spermatocytes is KLP3A, a kinesin-like proteinrequired for meiotic cytokinesis which localizes to the centralspindle midzone during anaphase and telophase (Williams etal., 1995). Similarly, the Pavarotti protein, a kinesin-likeprotein required for mitotic cytokinesis, concentrates in theinterphase nuclei of cellularized embryos (Adams et al.,1998). The results on anillin, KLP3A and PAV-KLP suggestthat a nuclear localization may be a common feature of manyproteins involved in cytokinesis. The confinement of theseproteins to the nucleus during interphase may serve to preventuntimely interactions with the cortical cytoplasm andmicrotubules (Field and Alberts, 1995).

We have shown that in S5 spermatocytes anillin concentratesin the nucleolus, to be released again in the nucleoplasm whenthis structure breaks down during the S6 stage. Anaccumulation of anillin into the nucleolus prior to cell divisionis not evident in spermatogonia and has not been previouslydescribed in somatic cells and female germ cells (Field andAlberts, 1995). Moreover, although KLP3A is localized inthe primary spermatocyte nucleus like anillin, it does notconcentrate into the nucleolus. The reasons why anillin isspecifically imported into the nucleolus of S5 spermatocytesare currently unknown. This further compartmentalization ofthe protein may reflect the necessity of sequestering anillin toavoid interactions with the complex intranuclear structures ofS5 primary spermatocytes (Bonaccorsi et al., 1988; Cenci etal., 1994). Alternatively, anillin may play an active, as yetunknown biological role, within the nucleolus.

There are two types of dividing cells that do not concentrateanillin in the nucleus: the preblastoderm embryonic cells (Fieldand Alberts, 1995) and the secondary spermatocytes (thisreport). Interestingly, these two cell types also fail toaccumulate KLP3A (Williams et al., 1995). The reasons for theexclusion of anillin from syncytial embryonic nuclei andsecondary spermatocyte nuclei are unclear. Field and Albertssuggested that anillin uptake in precellularization embryonicnuclei may be inactivated to allow anillin to interact with thecortical structures of the embryo. Clearly, this explanation doesnot apply to secondary spermatocytes which do not possessanillin-enriched cortical structures comparable to those presentin syncitial embryos. A common feature of embryonic cells



and secondary spermatocytes is the duration of interphase,which is much shorter than that of spermatogonia, primaryspermatocytes or postcellularization somatic cells (Karr andAlberts, 1986; Cenci et al., 1994). It is thus possible that therelative brevity of embryonic and secondary spermatocyteinterphases does not allow sufficient time for anillin to beimported into the nucleus.

Fusome morphogenesisThe analysis of testis preparations simultaneously stainedfor anillin and actin provided information on fusomemorphogenesis in Drosophila males. The fusome was firstdescribed by Giardina (1901) and subsequently found inseveral orders of insects (reviewed by Telfer, 1975; de Cuevaset al., 1997). In D. melanogaster females the fusome developsduring cystoblast divisions and eventually forms a branchedstructure that traverses each ring canal within stage 1 cysts(Storto and King, 1989; Lin and Spradling, 1995). Indirectimmunofluorescence studies have shown that the femalefusomes are highly enriched in α- and β-spectrin, in theadducin-like Hu-li tai shao (Hts) protein, in the Bag-of-marble(Bam) protein and in the cell cycle regulator cyclin A (Lin etal., 1994; Lin and Spradling, 1995; Mc Kearin and Ohlstein,1995; de Cuevas et al., 1996; de Cuevas and Spradling, 1998).Phalloidin staining revealed that female fusomes do not containdetectable amounts of F-actin (Warn et al., 1985; Spradling,1993).

Male fusomes, like their female counterparts, contain α-spectrin, the Hts and Bam proteins and cyclin A (Mc Kearinand Ohlstein, 1995; Eberhart et al., 1996; Giansanti et al.,1998). However, in contrast with oocyte fusomes, malefusomes are highly enriched in F-actin (Gunsalus et al., 1995;Hime et al., 1996). In the present study, we have examinedfusome morphogenesis during spermatogonial divisions and inboth spermatocyte and spermatid cysts. We have shown thatthe male fusome grows by a cyclic process of formation andfusion of fusome pieces. Nascent ring canals are progressivelyfilled up by fusome material approaching them from both sides.As cell cycle proceeds, this fusome plugs gradually fuse withthe preexisting central fusome, giving rise to a continuous,branched structure. This pattern of fusome growth isessentially identical to that recently described for the ovarianfusome (de Cuevas and Spradling, 1998). Thus, altough malesand females differ in both ring canal (Hime et al., 1996) andfusome composition, they maintain the same mechanism offusome development.

Our observations on fusome formation in Drosophilamales do not help to define the functional role of thisorganelle. Of the possible functions that have been proposedfor the fusome, two apply to the male germline (see Lin etal., 1994). First, the fusome may be required to control thepattern of germ cell interconnections by providing aframework for spindle orientation during gonial division.This is a likely hypothesis but certainly not exhaustive,because spermatid cysts which contain non-dividing cells,develop a highly branched fusome. Second, the fusome mayprovide a physical support for intercellular signaling. Wefavour this possibility, which we believe is in agreement withall the extant observations on both male and female fusomes.However, we would like to point out that there are no datawhich specifically support this conclusion. Nor there are

clues on the possible molecular basis for fusome-mediatedintercellular signaling.

The role of anillin during meiotic cytokinesisIt has been previously reported that during anaphase andtelophase anillin becomes highly enriched in the cleavagefurrow in a variety of Drosophila mitotic cells and in malemeiotic cells (Field and Alberts, 1995; Hime et al., 1996; deCuevas and Spradling, 1998). In the present study we haveexamined the relationships between anillin and the contractilering both in wild-type male meiosis and in meiotic divisionsof mutants defective in either contractile ring formation ordisassembly.

In wild type, anillin concentrates in a circumferential bandaround the equator of cells in meiotic anaphase, before theassembly of the F-actin-based contractile ring. A comparisonof anillin and tubulin immunostaining with the underlyingphase-contrast images, revealed that this anillin band is notassociated with the central spindle midzone but outlines thecell equator from mid-anaphase through telophase. Thisstaining pattern, which is particularly evident in cells fixed withformaldehyde prior to squashing, strongly suggests that anillinis bound to the equatorial cortex. Additional evidence thatanillin concentration in the cleavage furrow area does notdepend upon either the central spindle or the F-actin contractilering is provided by the cytological analysis of meiotic divisionsin chic and KLP3A mutants. Mutations in these loci suppressboth central spindle and contractile ring assembly, but do notaffect anillin concentration at the equator of the cell at mid-anaphase.

Although the initial formation of the anillin cortical banddoes not require the presence of an F-actin ring, thesestructures, once formed, precisely colocalize throughoutanaphase and telophase. In the absence of a contractile ring, asit happens in chic and KLP3A mutants, the anillin cortical banddoes not shrink as in wild type; it maintains its initial size and,in most cells, degenerates at the end of telophase. Thesefindings strongly suggest that the constriction of the anillinband is driven by the acto-myosin contractile ring.

Taken together, our results suggest a model for the role ofanillin during cytokinesis (Fig. 10). Anillin would firstconcentrate in the cleavage furrow area through an as yetunknown mechanism, where it would bind the equatorialcortex. This binding may be mediated by its carboxy-terminalpleckstrin homology (PH) domain (Straight et al., 1998); PHdomains are found in numerous membrane-associated proteinsand have been implicated in protein-protein and protein-phospholipid interactions (reviewed by Shaw, 1996). Oncebound to the equatorial cortex anillin would interact with theactin filaments of the contractile ring through its actin-bindingdomain, anchoring this structure to the plasma membranethroughout cytokinesis. With respect to the interactionbetween anillin and F-actin-based contractile ring, we canenvisage two possibilities. The assembly of the F-actin ringmay be independent of the anillin equatorial band, and theactin filaments would interact with anillin because of the closeproximity of these structures. Alternatively, it is conceivablethat the anillin band directs the formation of the F-actin ring,perhaps providing a framework for the correct assembly ofthis structure. At present we cannot discriminate betweenthese two alternatives. An understanding of the exact

2332 M. G. Giansanti, S. Bonaccorsi and M. Gatti

Fig. 8. Late telophases and spermatids in chic and tsrmutants. Testes, fixed according to protocol 1 (seeMaterials and Methods), were sequentially stained withanti-anillin antibody (red) and Hoechst 33258 (blue).(A,C,E) Late telophases I in wild-type (A), chic (C) andtsr (E) males. In the chic telophase (C) the anillin bandfailed to contract and is dislocated from the cell equator,whereas in the tsr telophase (E) the anillin ring isovercontracted with respect to wild type.(B,D,F) Spermatids in wild-type (B), chic (D) and tsr(F) males. In wild-type spermatids each nucleus isassociated with a single nebenkern (NK). (D) Twoabnormal chic spermatids resulting from failures incytokinesis; one of them has four nuclei associated witha large nebenkern (NK4), and the other is composed bytwo nuclei and one large nebenkern (NK2). The two tsrspermatids shown in F are also abnormal; they bothcontain two nuclei associated with a large nebenkern(NK2). The RCs are indicated by arrows; note that theRC in chic spermatids is larger, and those in tsrspermatids are smaller than wild-type RCs. Bar, 5 µm.

Fig. 7. Anillin behavior duringmeiosis of chic and KLP3Amutant males. Testispreparations were fixedaccording to protocol 3 (seeMaterials and Methods) andsequentially stained with anti-anillin (red) and anti-α tubulinantibodies, and with Hoechst33258 (blue). (A,C,E,G) Onlytubulin staining is shown;(B,D,F,H) Hoechst and anillinstainings are merged. (A,B) Awild-type late anaphase;(C,D) a chic late anaphase;(E,F) a wild-type telophase;(G,H) a KLP3A telophase.Note the absence of centralspindle in both chic andKLP3A ana-telophases, whichexhibit an unconstricted anillinequatorial band. Bar, 5 µm.


relationships between the anillin band and the F-actin ringmust await the phenotypical analysis of mutants in the anillingene.

We believe that the observations on tsr mutants are inagreement with the above model and provide an explanationfor the failure of cytokinesis frequently observed in thesemutants. In meiotic divisions of tsr males the equatorial bandof anillin and the actin ring colocalize during anaphase andearly telophase. However, when the actin ring overgrowsduring late telophase, the anillin band contracts further butmaintains a normal shape, and only partially colocalizes withthe abnormally large F-actin contractile structures. Theseresults strongly suggest that in tsr meiotic divisions anillinnormally mediates the anchoring of the contractile ring to theplasma membrane. When the actin ring overgrows, due to thereduced cofilin activity, the anillin circumferential bandbecomes overcontracted but maintains an overall normal

structure. It is conceivable that the F-actin overgrowth canresult in a detachment of the plasma membrane from the anillinring, thus impairing the cytokinetic process (Figs 9 and 10).

We thank Chris Field for the anillin antibodies, and Anna Bodinifor the artwork. This work was supported in part by a TMR grant ofthe EU.

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Fig. 10. A model for the role of anillin during meiotic cytokinesis.The plasma membrane is depicted in black, anillin in green, actin inred, and chromatin in blue. (A) A mid-anaphase (stage M4b); (B)late anaphase (stage M4c); (C) telophase (stage M5). In wild-typemid-anaphase, anillin concentrates in a narrow equatorial band andbinds the plasma membrane prior to the assembly of the contractilering. In late anaphase the anillin band interacts with the actomyosinring, anchoring it to the cell cortex. Anillin remains intimatelyassociated with the cytokinetic ring throughout telophase andcontracts along with it, thus mediating membrane invagination. Inchic mutants, anillin concentration in the cleavage furrow occursnormally, but the anillin band fails to contract and eventuallydegenerates, due to the absence of the actomyosin ring. In tsrmutants anillin behavior is regular till late telophase, when thecytokinetic ring fails to disassemble and overgrows. Thisphenomenon leads to an overcontraction of the anillin band and mayresult in its detachment from the plasma membrane, with aconsequent impairment of cytokinesis. See text for a more detailedexplanation.

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