INTRODUCTION

Accurate chromosome segregation is essential for thepropagation of cell life. In eukaryotic cells, chromosomesegregation is dependent on the assembly and function of acomplex, transient, and dynamic structure known as themeiotic or mitotic spindle. The most common form of spindlefound in eukaryotic cells is bipolar. Bipolar spindle-chromosome-aster complexes generally contain two asters, oneat each pole, each of which contains a centrosome thatnucleates and organizes a radial array of microtubules (MTs;Wilson, 1925; Mazia and Dan 1952; Schrader, 1953; Inoue,1964, 1981; Inoue and Sato, 1967; Picket-Heaps et al., 1982;Kubai, 1975; Heath, 1980; Mitchison, 1989; McIntosh andKoonce, 1989). However, there are numerous examples ofalternative forms of spindle structure (Waters and Salmon,1997). For example, cells can assemble spindles which containonly a single pole (monopolar or monastral spindles) (Maziaet al., 1960; Gerbi, 1986; Ito et al., 1994). Still others containno asters at all, yet can organize MTs into bipolar arrangements(acentrosomal or anastral spindles) (Bajer and Mole-Bajer,

1982; Bastmeyer et al., 1986; Steffen et al., 1986; McKim andHawley, 1995; Theurkauf and Hawley, 1992; Albertson andThomson, 1993; Heald et al., 1996, 1997). Importantly, bothmonastral and anastral spindles segregate chromosomesefficiently.

It has long been thought that as major microtubuleorganizing centers (MTOCs), centrosomes play a key role inbipolar spindle assembly (McIntosh, 1983; Sluder and Rieder,1985; Kirschner and Mitchison, 1986; Mazia, 1987; Rieder andAlexander, 1990; Zhang and Nicklas, 1995; Mitchison, 1989;Rieder, 1991). Thus, it has been proposed that in animal cells,during a typical cell division cycle, dynamic MTs emanatingfrom duplicated centrosomes capture the kinetochores that arelocated on sister chromosomes and, through the additionalaction of as yet undefined forces, bipolar spindles areestablished (Kirschner and Mitchison, 1986). However, aconsiderable body of evidence suggests that the MTcytoskeleton can reorganize to exhibit ‘polarity’ even in theabsence of centrosomes (McNiven et al., 1984; Rodionov andBorisy, 1997; Heald et al., 1997; Merdes and Cleveland, 1997;Compton, 1998), bringing to question the role, if any, of the

1291Journal of Cell Science 112, 1291-1302 (1999)Printed in Great Britain © The Company of Biologists Limited 1999JCS0143

Meiosis I spindle assembly is induced in lysate-extractmixtures prepared from clam (Spisula solidissima) oocytes.Unactivated lysate prepared from unactivated oocytescontain nuclei (germinal vesicles, GVs) which housecondensed chromosomes. Treatment of unactivated lysatewith clarified activated extract prepared from oocytesinduced to complete meiosis by treatment with KCl inducesGV breakdown (GVBD) and assembly of monopolar,bipolar, and multipolar aster-chromosome complexes. Theprocess of in vitro meiosis I spindle assembly involves theassembly of microtubule asters and the association of theseasters with the surfaces of the GVs, followed by GVBD andspindle assembly. Monoclonal antibody m74-1, known toreact specifically with the N terminus of the intermediatechain of cytoplasmic dynein, recognizes Spisula oocyte

dynein and inhibits in vitro meiosis I spindle assembly.Control antibody has no affect on spindle assembly. Asimilar inhibitory effect on spindle assembly was observedin the presence of orthovanadate, a known inhibitor ofdynein ATPase activity. Neither m74-1 nor orthovanadatehas any obvious affect on GVBD or aster formation. Wepropose that dynein function is required for the associationof chromosomes with astral microtubules during in vitromeiosis I spindle assembly in these lysate-extract mixtures.However, we conclude that dynein function is not requiredfor centrosome assembly and maturation or forcentrosome-dependent aster formation.

Key words: Spisula, Centrosome, Dynein, Meiosis, Mitosis

SUMMARY

Dynein is required for spindle assembly in cytoplasmic extracts of Spisula

solidissima oocytes

Robert E. Palazzo1,2,*, Eugeni A. Vaisberg2,3, Dieter G. Weiss4, Sergei A. Kuznetsov2,4 and Walter Steffen2,5

1The Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66045, USA2The Marine Biological Laboratory, Woods Hole, MA 02543, USA3The Department of Molecular, Cell and Developmental Biology, University of Colorado, Boulder, CO 80309-0347, USA4Institute of Zoology, University of Rostock, D-18051 Rostock, Germany5Institute of Biochemistry and Molecular Cell Biology, Biocenter, University of Vienna, A-1030 Vienna, Austria*Author for correspondence (e-mail: palazzo@aster.bio.ukans.edu)

Accepted 11 February; published on WWW 8 April 1999

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centrosome in bipolar spindle assembly (Heald et al., 1996,1997; Gaglio et al., 1996, 1997; Bonaccorsi et al., 1998; deSaint Phalle and Sullivan, 1998).

Clearly, understanding the forces and the dynamic molecularinteractions that contribute to the spatial and temporalorganization of the MT cytoskeleton during spindle assemblyis a formidable challenge. Such a pursuit would benefit from,and may ultimately require, the development of functionalreconstitution systems for the study of spindle assembly underdefined conditions. The possibility for developing suchsystems was encouraged by the demonstration thatconcentrated lysates prepared from Xenopus laevis oocytes arecapable of executing numerous cell cycle-dependent events,including spindle assembly and function, outside the confinesof a living cell (Lohka and Mahler, 1985; Murray, 1991; Sawinand Mitchison, 1991; Sawin et al., 1992; Murray et al., 1996).Introduction of Xenopus sperm nuclei into these oocyte lysatesresults in the formation of monopolar, bipolar and multipolarspindles (Lohka and Mahler, 1985; Sawin and Mitchison,1991). Surprisingly, introduction of DNA-coated magneticbeads into these lysates also results in the formation of bipolarspindles (bead-spindles) even in the absence of centrosomesand centromeric DNA, and therefore, presumably kinetochores(Heald et al., 1996). Importantly, assembly of these variousspindle forms in Xenopus oocyte lysates is dependent on forcesgenerated by MT-dependent motor proteins (Sawin et al., 1992;Heald et al., 1996).

Here we describe a cell-free system that is capable ofexecuting meiosis I spindle assembly in vitro. We show thataster-chromosome complexes, including bipolar spindles, canbe assembled in cytoplasmic extracts prepared from Spisulasolidissima oocytes. The Spisula oocyte system offers anumber of unique advantages for the study of spindleassembly. First, oocytes can be obtained in large (100 g)quantities on a daily basis when animals are gravid, making abiochemical approach to understanding spindle assemblypossible. Second, the oocytes are obtained naturally arrestedat one specific phase of the meiotic cell cycle, late prophaseof meiosis I, and contain germinal vesicles (GVs) which housecondensed chromosomes (Allen, 1953; Longo and Anderson,1970; Dessev and Goldman, 1988; Dessev et al., 1989;Ruderman et al., 1997). Thus, Spisula oocytes are poisedprecisely at the G2/M border of meiosis I, and can be inducedto cross that border, break down their GVs, and assemblespindles within 15 minutes by artificial activation with KCl(Allen, 1953; Rebhun 1959). Thus, KCl activation offers aconvenient method for induction of the synchronous assemblyof bipolar meiosis I spindles in a large population of cells(Allen, 1953; Rebhun, 1959; Rebhun and Sharpless, 1964).Importantly, we and others, have demonstrated thatcytoplasmic extracts prepared from Spisula oocytes arecapable of executing numerous complex cell cycle-dependentevents including spontaneous aster formation (Weisenberg andRosenfeld, 1975; Palazzo et al., 1988, 1992), nuclear envelope(GV) breakdown (Dessev et al., 1989, 1991), centrosomematuration and centriole duplication (Palazzo et al., 1992),and the regulated proteolysis of cyclin proteins (Ruderman etal., 1997). Finally, methods have been developed for theisolation of nuclei (GVs) (Dessev et al., 1989), centrosomes(Vogel et al., 1997; Palazzo and Vogel, 1999), asters (Palazzoet al., 1988), microtubule proteins (Palazzo et al., 1988;

Suprenant, 1991), and enzymes known to be involved in cellcycle regulation (Ruderman et al., 1997). Thus, with thissystem, the goal of spindle assembly under defined conditionsis a real possibility.

A number of recent studies have indicated that theinteraction of MTs with MT-dependent motor proteins iscrucial for the temporal and spatial organization of complexcytoskeletal structures in living cells (Rodionov and Borisy,1997; Inoue et al., 1998), including bipolar spindles (Vaisberget al., 1993; Matheis et al., 1996; Gaglio et al., 1996, 1997). Inparticular, substantial evidence gained from studies in anumber of different experimental systems indicates that themotor protein dynein is required for aster formation (Verde etal., 1991; Gaglio et al., 1997; Inoue et al., 1998) and spindleassembly (Pfarr et al., 1990; Steuer et al., 1990; Vaisberg et al.,1993; Merdes et al., 1996; Gaglio et al., 1996, 1997). Further,addition of dynein antibodies to Xenopus extracts inhibits bothsperm-spindle assembly and bead-spindle pole formation,indicating that dynein is required for both the assembly andstability of spindle poles in anastral spindles assembled in vitro(Heald et al., 1996, 1997). Therefore, we used the in vitroSpisula spindle assembly assay described to test the role ofdynein in both aster formation and spindle assembly. We showthat a monoclonal antibody raised against the intermediatechain of cytoplasmic dynein inhibits spindle assembly, but hasno affect on aster formation.

MATERIALS AND METHODS

Oocyte lysatesMature surf clams, Spisula solidissima, were obtained from theMarine Biological Laboratory, Woods Hole, MA. Gonads weredissected from mature females, minced, and resuspended in filteredsea water. Released oocytes were poured through cheesecloth toremove gonadal tissue, and oocytes were washed with filtered seawater by successive cycles of resuspension and settling (Allen, 1953;Rebhun and Sharpless, 1964; Costello and Henley, 1971; Palazzo etal., 1988). Washed oocytes remain arrested in late prophase of meiosisI until fertilized or artificially activated with KCl to induce thecompletion of meiosis (Allen, 1953). Lysates were prepared fromeither quiescent, unactivated oocytes, or activated oocytes fourminutes after KCl treatment (Palazzo et al., 1988; Vogel et al., 1997;Palazzo and Vogel, 1999). Centrosome-free high-speed-activated-extract (activated extract), was prepared from activated oocytesaccording to the method of Palazzo et al., (1992) and kept on ice untiluse.

For preparation of unactivated oocyte lysate (unactivated lysate), 10ml of a 10% oocyte/sea water suspension was centrifuged at 1,000 gfor 1.0 minute. The oocyte pellet was aspirated dry, quicklyresuspended in 10-12 volumes of 1.0 M glycerol, and centrifugedagain. Pellets were aspirated dry, resuspended in 10-12 volumes ofglycerol-phosphate buffer (1.0 M glycerol, 10 mM NaH2PO4, pH 8.0),incubated at room temperature for 1.0 minute, and centrifuged. Thepellet was aspirated dry, quickly resuspended in aster buffer (20 mMPipes, 100 mM NaCl, 5 mM MgSO4, pH 7.2) and centrifuged.Oocytes will lyse in aster buffer, so it was important to proceedthrough this step quickly. The pellet was again aspirated dry, and theoocytes lysed by gentle vortexing or, preferably, by flicking the tubevigorously by hand. All solutions and procedures for preparation ofunactivated lysate were at room temperature. Oocyte lysis wasmonitored by viewing with a phase-contrast microscope. Typically,unactivated lysate contained an abundance of free GVs, but no intactoocytes. GVs remained intact in unactivated lysate for hours at room

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1293Dynein in spindle assembly in Spisula solidissima oocytes

temperature. Following oocyte lysis, unactivated lysate was placed onice until use.

In vitro spindle assembly assayIn vitro spindle assembly was induced by gently mixing equalvolumes of activated extract and unactivated lysate on ice. Sampleswere warmed to room temperature and at various time points, 10 µlaliquots were removed, diluted into 100 µl of microtubule stabilizingbuffer (MSB: 20 mM MES, 10 mM EGTA, 5 mM MgSO4, and 20%glycerol, pH 6.3-6.5) containing 1% Triton X-100 or Nonidet P-40(lysis buffer), and samples quickly fixed by addition of 3.0 ml of PBS(pH 7.2) containing 0.5% glutaraldehyde and 0.4% Triton X-100.Samples were centrifuged onto polylysine coated coverslips at 12,000g, and processed for immunofluorescence microscopy by amodification of the methods of Mitchison and Kirschner (1984, 1986)as previously described (Palazzo et al., 1992; Vogel et al., 1997;Palazzo and Vogel, 1999). In addition, chromosomes were stained byaddition of Hoechst 33342 during one of the wash steps.

Inhibition of spindle assemblyFor experiments involving vanadate, activated extract and unactivatedlysate were each supplemented with 0 µM, 2.0 µM or 10.0 µMvanadate, and incubated on ice for 10 minutes before use. Forexperiments involving antibodies, activated extract and unactivatedlysate were each supplemented with either 0.5 mg/ml of Protein Gpurified dynein IC specific antibody, m74-1, or Protein G purifiedcontrol antibody and incubated on ice for 15 minutes. Treated extracts(activated extract and unactivated lysate) were mixed and warmed toroom temperature. After incubation for 15 to 20 minutes, sampleswere resuspended in lysis buffer to terminate the reactions andanalyzed by DIC-microscopy. In addition, to visualize spindlemicrotubules and chromosomes by fluorescence microscopy, lysate-extract mixtures were supplemented with 0.2 to 0.5 mg/ml rhodamine-labelled tubulin (see below) while incubating on ice. Chromosomeswere stained by adding 1 µg/ml Hoechst 33258 to the lysis buffer.

AntibodiesMonoclonal antibody m74-1 specific for the N terminus of dynein IChas been characterized previously (Steffen et al., 1997). Monoclonalantibody m150-1 was developed against dynactin p150Glued (Steffenet al., 1996, 1997). This antibody recognizes p150Glued in variousvertebrate cells; however, it does not affect the function of cytoplasmicdynein (W. Steffen, unpublished data). Tubulin monoclonal antibody(Tu27; generous gift of Dr Anthony Frankfurter) and rhodaminesecondary antibodies (Calbiochem) were used to stain MTs.

DIC and fluorescence microscopySamples were observed by video enhanced contrast, differentialinterference contrast (DIC) microscopy (Allen et al., 1981, 1985;Weiss et al., 1989) and video intensified fluorescence microscopy(Weiss et al., 1989). A Zeiss Axiophot microscope (C. Zeiss, Inc.,Thornwood, NY) equipped with oil immersion condenser, ×40 and×100 DIC Plan Neofluar oil objectives was used. The mercury arclamp (HBO 100) for DIC was modified to generate full condenseraperture illumination by the addition of an Ellis fiberoptic lightscrambler (Technical Video, Ltd, Woods Hole, MA). A HamamatsuC2400-07 Newvicon camera was used to acquire DIC images and aHamamatsu C2400-97 Intensified CCD camera system (HamamatsuPhotonics, Inc., Bridgewater, NJ) was used to acquire fluorescenceimages. The analog and digital processing of fluorescence and DICsignals was performed in parallel using two ARGUS 10 real timeimage processors (Hamamatsu Photonics Inc.). A Sony SVHS videorecorder was used to record processed images. Single frame imageswere captured from video tape or directly from ARGUS 10 imageprocessors using a Power MacIntosh 7500 (Apple Computer,Cupertino, CA) equipped with a LG-3 frame grabber (ScionCorporation, Frederick, MD) and NIH Image software. In addition,

double stained fluorescence samples were photographed by doubleexposure of Kodak Ektachrome 400 slide film and images weredigitized by scanning the original slide film using a Nikon (NikonGmbH, Düsseldorf, Germany) or a Polaroid SprintScan 35 (Polaroid).Final figures were prepared using Adobe Photoshop software.

ImmunoprecipitationCytoplasmic extracts of Spisula oocytes were diluted 5-fold withTBS-I (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM DTT plusprotease inhibitors 10 µg/ml leupeptin, 10 µg/ml pepstatin, 1 mMPMSF, and 10 µg/ml TAME) and then centrifuged at 100,000 g for10 minutes. The supernatant was then supplemented with 1% TritonX-100 and again centrifuged at 100,000 g for 30 minutes. Supernatantwas supplemented with 0.1 mg/ml control antibody m150-1 or dyneinIC specific antibody m74-1 and incubated for 4 hours at 4°C.Immunoprecipitation was carried out by adding 10% v/v of a 10%w/v Protein A-Sephadex CL-4B (Pharmacia, Uppsala, Sweden) inTBS-I buffer containing 2 mg/ml bovine serum albumin. Afterincubation for 90 minutes at 4°C, Sepharose beads were washed 5-times with TBS-I containing 1% Triton X-100 and 3-times with TBS-I. Bead pellets were extracted with Laemmli buffer and then analyzedby SDS-PAGE and immunoblot.

SDS-PAGE and immunoblotSDS-PAGE was carried out according to the method of Laemmli(1970). Electrotransfer to nitrocellulose was carried out according tothe method of Towbin et al. (1979) with some modification asdescribed earlier (Steffen et al., 1997). Antigens were detected with aperoxidase-conjugated goat anti-mouse antibody (Bio-Rad,Richmond, CA) and visualized using a luminescence assay (Pierce,Rockford, IL).

Rhodamine-labelled tubulinTubulin was isolated from bovine brain and purified as describedpreviously (Steffen et al., 1997). DEAE-purified tubulin was labeledwith 5,(and-6)-carboxytetramethyl-rhodamine succinimide ester(CTMR) (Molecular Probes, Eugene, Oregon) according to a protocolof the laboratory of Mitchison (Hyman et al., 1991;http://skye.med.harward.edu/Protocols/mt3.html). Tubulin waspolymerized at 37°C by adding 1 mM GTP and 10%dimethylsulfoxide (DMSO) and the microtubules were centrifugedthrough a high pH glycerol cushion (100 mM Na-HEPES, pH 8.6, 1mM MgCl2, 1 mM EGTA, 60% glycerol). The microtubule pellet wasresuspended in labeling buffer and labeled by the addition of 10 mMCTMR. The yield of label was determined to be approximately 0.7mol CTMR per mol of monomeric tubulin. Rhodamine-labelledtubulin was resuspended in 50 mM K-glutamate containing 0.5 mMMgCl2. The protein concentration was adjusted to 10 mg/ml. Thelabeled tubulin was frozen as 5 µl aliquots in liquid N2 and stored at−80°C.

RESULTS

In vitro spindle assemblySpisula oocytes remain arrested in late prophase at the G2/Mborder of meiosis I until fertilized or artificially activated(Allen, 1953; Rebhun, 1959; Dessev and Goldman, 1988).Unactivated oocytes contain a germinal vesicle (GV) whichhouses chromosomes, however, no centrosomes, asters, orpolymerized MTs have been detected in these oocytes by lightmicroscopy (Allen, 1953; Rebhun, 1959), electron microscopy(Longo and Anderson, 1970; Sachs, 1971) orimmunofluorescence microscopy using tubulin antibodies(Kuriyama et al., 1986). Artificial activation of Spisula oocytes

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with KCl induces GV breakdown (Dessev and Goldman,1988), maternal centrosome maturation (Palazzo et al., 1992)characterized by centriole duplication and a time-dependentincrease in the centrosomes microtubule nucleation potential,and assembly of meiosis I spindles within 15 minutes (Allen,1953; Rebhun, 1959; Longo and Anderson, 1970; Sachs, 1971;Kuriyama et al., 1986).

Previously, we reported that treatment of lysate preparedfrom unactivated oocytes with centrosome-free extract(activated extract) prepared from Spisula oocytes 4 minutesafter KCl activation, induces the maturation of centrosomes invitro, with fidelity to the in vivo centrosome maturation process(Palazzo et al., 1992). We extended this analysis to determineif GV breakdown and in vitro spindle assembly might also beoccurring in these lysate-extract mixtures. Thus, unactivatedlysate was treated with equal volume of activated extract andmixtures were observed by DIC microscopy while warming toroom temperature (Fig. 1). Initially, lysate-extract mixturescontained an abundance of GVs. However, within 4 minutesafter warming, the outline of the GVs became less distinct. By8 minutes after warming GVs were difficult to discern, and by12 minutes they had completely disappeared.

To extend this analysis further, activated extract (Fig. 2A)unactivated lysate (Fig. 2B) and unactivated lysate treated withactivated extract (Fig. 2C) were incubated at room temperaturefor 16 minutes, fixed, centrifuged onto glass coverslips andprepared for double label immunofluorescence microscopy(Palazzo et al., 1992) using Hoechst 33342 to labelchromosomes and tubulin antibody to label MTs. Nochromosomes, asters or aster-like structures could be identifiedin activated extract (Fig. 2A) indicating the absence ofcentrosomes in this fraction. As expected from the previousexperiment, unactivated lysates contained an abundance ofintact GVs which housed chromosomes (Fig. 2B). In addition,a complex MT array composed of many long microtubules wasalso present in the unactivated lysate (Fig. 2B). Regardless ofthe presence of these MT arrays, the results indicate that thepreparation of unactivated lysate did not activate the cell cycle-dependent machinery that is required to induce GV breakdown

(GVBD) or spindle assembly. However, no intact GVs and noMT networks were found in unactivated lysate that had beentreated with activated extract (Fig. 2C). Instead, these lysate-extract mixtures contained numerous monopolar, bipolar andmultipolar aster-chromosome complexes (Fig. 2C). Nochromosomes or asters were found in activated extract at anytime (Fig. 2A), indicating that structural components withinunactivated lysate were necessary for spindle assembly.

To determine if the bipolar aster-chromosome complexesidentified were representative of bipolar spindle assembly inthese lysate mixtures, and not the result of centrifugationduring sample preparation, unactivated lysate treated withactivated extract was warmed to room temperature for 12minutes and diluted into microtubule stabilization buffersupplemented with Triton X-100 detergent to solubilizemembrane and yolk granules and to clarify the solution.Observation with DIC microscopy revealed the presence ofbipolar spindles in these diluted lysate-extract mixtures. Mostof these spindles contained chromosomes aligned in typicalmetaphase arrays (Fig. 3A and B). However, in someexperiments, spindles were also found which containedchromosomes in what appeared to be anaphase arrays (Fig. 3Cand D). These results indicate that treatment of unactivatedlysate with activated extract induced GV breakdown andspindle assembly within the time frame expected for spindleassembly in intact oocytes.

Time course of in vitro spindle assemblyTo examine the sequence of events leading to in vitro spindleassembly, cell extracts were analyzed at various time pointsafter warming to room temperature. Thus, unactivated lysatesand unactivated lysates treated with activated extracts weremixed on ice, warmed to room temperature and aliquots wereremoved at 0, 4, 8 and 16 minutes and prepared forimmunofluorescence microscopy. As expected, throughoutthis time course, unactivated lysate contained an abundance ofGVs with associated chromosomes (Fig. 4A-D). Interestingly,small MT asters were identified in unactivated lysate 0minutes after warming to room temperature (Fig. 4A). By 4

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Fig. 1. Germinal vesicle breakdown in vitro.Unactivated lysate treated with activatedextract was placed in a microscopic flowchamber and incubated at room temperature.DIC images of the same area were takenafter 0, 4, 8, and 12 minutes of incubation.Lysate-extract mixtures contain GVs(arrows) which begin to loose distinction by4 minutes. By 8 minutes GVs are difficult todiscern, and by 12 minutes they havedisappeared. Bar, 20 µm.

1295Dynein in spindle assembly in Spisula solidissima oocytes

minutes after warming, MTs associated with these asters hadgrown longer (Fig. 4B), and by 8-16 minutes after warming,unactivated lysate contained such an extensive network ofMTs that these astral centers could no longer be distinguished(Fig. 4C and D). Thus, in contrast to previous studies, theseresults suggest that unactivated oocytes may contain acentrosome precursor or ‘procentrosome’ as originallysuggested by Weisenberg and Rosenfeld (1975) which iscapable of nucleating very few microtubules but has escapedprevious detection in unactivated oocytes (Kuriyama et al.,1986; Longo and Anderson, 1970; Sachs, 1971) and

unactivated oocyte lysates (Weisenberg and Rosenfeld, 1975;Palazzo et al., 1992).

Time course analysis of unactivated lysate treated withactivated extract revealed that asters within these lysate-extractmixtures undergo a time-dependent increase in size (Fig. 4E-H), verifying previous results (Palazzo et al., 1992). Smallasters were found in lysate-extract mixtures 0 minutes afterwarming to room temperature, but by 4-8 minutes afterwarming, asters had grown in size and were often foundassociated with the surfaces of deformed GVs (Fig. 4F and G).By 16 minutes, no GVs could be identified in these lysate-extract mixtures. Instead, an abundance of aster-chromosomecomplexes were found (Fig. 4H). These results indicate thattreatment of unactivated lysate with activated extract inducesGV breakdown (GVBD) and assembly of aster-chromosomecomplexes reflective of the events that lead to meiosis I spindleassembly in living oocytes.

Vanadate inhibits spindle assembly but not asterformationSeveral studies have indicated that cytoplasmic dynein plays acritical role in the assembly of asters and spindles (Vaisberg etal., 1993; Gaglio et al., 1996, 1997; Merdes et al., 1996; Healdet al., 1997; Inoue et al., 1998). The development of the Spisulaspindle assembly assay described here offered the opportunityto test the role of dynein in the in vitro assembly of meiosis Ispindles. Thus, we tested the ability of vanadate, a knowninhibitor of dynein ATPase activity (Gibbons et al., 1978;Shpetner et al., 1988), to inhibit in vitro spindle assembly.Unactivated lysate and activated extract were supplementedwith 0 µM, 2 µM or 10 µM vanadate, mixed and warmed toroom temperature for 18 minutes. Samples were then dilutedin lysis buffer and examined by fluorescence microscopy.

For each vanadate concentration tested, 25 microscope fieldswere chosen and observed randomly, and the number of asterswith attached chromosomes were counted. In the presence orabsence of vanadate, no difference in GVBD or aster formationwas observed. However, in the presence of vanadate, thenumber of asters with attached chromosomes was dramaticallyreduced in a concentration-dependent manner (Table 1). At aconcentration of 10 µm vanadate, a 71% inhibition of theformation of aster-chromosome complexes was observed.Since vanadate is a specific inhibitor of dynein ATPase activityat the concentrations used (Gibbons et al., 1978; Shpetner etal., 1988; also see Porter et al., 1987), these results suggestedthat dynein function is necessary for chromosome attachmentto astral microtubules, but not for aster formation.

Monoclonal antibody m74-1 recognizes cytoplasmicdynein in Spisula oocytesTo extend the analysis of the role of dynein in in vitro spindleassembly, we tested a dynein-specific monoclonal antibody,m74-1, for the ability to bind Spisula dynein components andinhibit in vitro spindle assembly (see below). We havepreviously demonstrated that antibody m74-1 reactsspecifically with the N terminus of the intermediate chain ofcytoplasmic dynein (Steffen et al., 1997). Furthermore, wedemonstrated that antibody m74-1 interferes with theinteraction of cytoplasmic dynein with dynactin, therebyblocking the function of cytoplasmic dynein in the MT-dependent transport of membranous organelles.

Fig. 2. In vitro assembly of aster-chromosome complexes.Immunofluorescence microscopy of activated extract (A),unactivated lysate (B), and unactivated lysate treated with activatedextract (C), warmed to room temperature for 16 minutes. Nochromosomes (blue), asters or microtubules (red) are found inactivated extract (A). Unactivated lysate contains GVs which housecondensed chromosomes (arrowheads in B) and an elaborate matrixof polymerized microtubules (B). Lysate-extract mixture containsaster-chromosome complexes, but no intact GVs and no microtubulematrix (C). Bar, 25 µm.

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SDS-PAGE and immunoblot analysis revealed thatmonoclonal antibody m74-1 recognizes two polypeptides ofapproximately 80- and 160 kDa present in Spisula oocytes(Fig. 5A). The 80 kDa polypeptide probably represents theintermediate chain of cytoplasmic dynein, while the nature ofthe 160 kDa band is not yet clear. To further characterize them74-1 related antigen, monoclonal antibody m74-1 was usedto immuno-precipitate the antigen from oocyte extracts. Aprotein complex was isolated which contained one highmolecular mass component, in addition to several intermediateand low molecular mass components (Fig. 5B). Again, both the80 kDa and 160 kDa polypeptide bands were detected whenthe immuno-precipitated material was analyzed byimmunoblot. These results strongly suggest that themonoclonal antibody m74-1 recognizes Spisula oocytecytoplasmic dynein.

Dynein antibody inhibits spindle formation but notaster formationWe used the m74-1 antibody to test if the function ofcytoplasmic dynein is necessary for spindle assembly in

Spisula oocyte lysate-extract mixtures. In addition, rhodamine-labelled tubulin and Hoechst 33342 were added to the mixturesto allow visualization of MTs and chromosomes. Whenunactivated lysate and activated extract were supplementedwith a control antibody (m150-1), mixed and warmed to roomtemperature, GV breakdown occurred normally within theexpected 8 minutes (data not shown). Importantly, by 20minutes after warming, spindles had clearly assembled in thepresence of control of antibody (Fig. 6A-D), verifying theresults of the DIC and immunofluorescence studies describedearlier. Thus, the addition of labeled tubulin and controlantibody had no affect on in vitro spindle assembly, and thepresence of meiotic spindles could easily be identified by thepresence of chromosome clusters using Hoechst DNA stain(Fig. 6A and A′).

When unactivated lysate and activated extract were mixedand incubated in the presence of dynein IC specific antibodym74-1, GV breakdown occurred normally (Fig. 6). However,in contrast to the effect of control antibody, spindle assemblywas completely inhibited by m74-1 (Fig. 6E-G). Unlike controlsamples, no chromosome clusters could be observed in m74-1treated extracts (Fig. 6E′-G). Importantly, although spindleassembly was inhibited, aster assembly appeared normal in thepresence of m74.1 antibody (Fig. 6E-G). Finally, no aster-chromosome complexes could be identified within lysate-extract mixtures which had been treated with antibody m74-1.Thus, although antibody m74-1 clearly inhibited in vitrospindle assembly, it had no affect on either GV breakdown oraster formation, suggesting that the m74-1 antibodyspecifically inhibited the interaction of chromosomes withastral MTs. Further, these results suggest that dynein function

R. E. Palazzo and others

Table 1. Effect of vanadate on number of asters withattached chromosomes

Vanadate concentration (µM)

0 2 10

Aster-chromosome complexes 37 24 11% Inhibition 0 27 71

The number of asters with attached chromosomes were scored within 25fields selected at random.

Fig. 3. Bipolar spindles assembled inlysate-extract mixture. DIC microscopy ofbipolar spindles isolated 12 minutes afterwarming lysate-extract mixture to roomtemperature. Spindles containchromosomes (arrows) aggregatedbetween two asters (arrowheads) in ametaphase arrangement (A and B). Somespindles appear to contain chromosomesin an anaphase arrangement (C and D).Bar, 20 µm.

1297Dynein in spindle assembly in Spisula solidissima oocytes

is not necessary for the process of centrosome maturationwhich is characterized by a time-dependent increase in the MTcontent of asters during meiosis I in Spisula oocytes (Palazzoet al., 1992).

DISCUSSION

Spindle assembly in Spisula oocyte extractsThe present study demonstrates that meiosis I spindle assemblycan be induced in lysate-extract mixtures prepared fromSpisula solidissima oocytes. Treatment of unactivated oocytelysates with a high-speed supernatant prepared from activated

oocytes (activated extracts) induces GV breakdown, asterformation, and assembly of monopolar, bipolar and multipolarspindles. Many of the bipolar spindles formed in these lysatemixtures appear to be reflective of the meiosis I spindles thatare assembled in living oocytes following KCl activation. Thetiming and the sequence of events leading to meiosis I spindleassembly appear to be similar if not identical, in vitro and inliving oocytes.

In living oocytes, approximately four minutes after KClactivation, two maternal asters appear in unison (Allen, 1953;Rebhun, 1959). During this time, the GV remains intact.However, by 8-10 minutes after oocyte activation, GVs beginto deform and breakdown, and asters, which become more

Fig. 4. Time course of in vitro spindleassembly. Unactivated lysate (A-D) andunactivated lysate treated with activatedextract (E-H) were warmed to roomtemperature and processed forimmunofluorescence microscopy(chromosomes, blue; microtubules, red)0 minutes (A and E), 4 minutes (B andF), 8 minutes (C and G) and 16 minutes(D and H) after warming to roomtemperature. At 0 minutes, unactivatedlysate (A) contains intact GVs and smallasters (arrowheads) which contain fewmicrotubules. By 4 minutes (B),microtubules have elongated from theseasters (arrowheads in B), and by 8minutes (C) unactivated lysates haveassembled a dense matrix ofmicrotubules. GVs remain intact inunactivated lysate throughout this period(A-D). Asters are present in lysate-extract mixtures 4 minutes afterwarming (F) and by 4-8 minutes astersare found associated with the surface ofGVs (arrows in F and G). Lysate-extractmixtures contain aster-chromosomecomplexes 16 minutes after warming(H), but no GVs and no microtubulematrix. Bar, 25 µm.

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prominent as a result of centrosome maturation and increasedmicrotubule content, are typically found associated with theGV surface (Allen, 1953; Rebhun, 1959). Finally, by 15minutes after activation, GVs have disappeared and bipolarmeiosis I spindle assembly is completed (Allen, 1953; Rebhun,1959; Rebhun and Sharpless, 1964). Following meiosis Ispindle assembly, KCl activated oocytes continue through themeiotic cell cycle, complete meiosis I and II, and enter themitotic cycle (Kuriyama et al., 1986). However, artificiallyactivated oocytes assemble monopolar spindles during themitotic cycle (Rebhun and Sharpless, 1964; Kuriyama et al.,1986) and are thus incapable of mitotic cleavage.

The in vitro assembly of Spisula spindles as described hereis clearly reflective of meiosis I spindle assembly in KClactivated oocytes: asters appear within four minutes of mixinglysate; asters grow in microtubule content and many are foundassociated with the surfaces of GVs by 8 minutes; and GVbreakdown and assembly of aster-chromosome complexes iscompleted by 15-16 minutes. Although monopolar and bipolarspindle structures can be found 15 minutes after mixingunactivated lysate with activated extracts, the majority of aster-chromosome complexes assembled in vitro were multipolar.Observation of aster-chromosome complexes which containedten or more asters was not uncommon in these lysate-extractmixtures. Clusters of chromosomes, reflective of congressedmetaphase chromosomes, were usually found aggregatedbetween any two asters within these multipolar complexes. Thein vitro formation of multipolar aster-chromosome complexes,which are never found in KCl activated oocytes (Palazzo,unpublished data), can simply be explained by the fact thatcentrosomes and GVs within the lysate-extract mixtures are notbounded by a plasma membrane. Thus, during aster formationand GV breakdown, a single aster can access more than one

GV as was observed (Fig. 4G). Unlike intact ooyctes whichcontain only two maternal asters, each capable of interactingwith a single GV during meiosis I, lysate-extract mixturescontain a plethora of asters and GVs, all capable of interactionswhich are only limited by their vicinity within the mixture.Thus, multipolar aster-chromosome complexes are expected toform under these conditions.

Comparison of in vitro spindle assemblymechanismsThe sequence of events during in vitro spindle assembly inSpisula oocyte lysate-extract mixtures differs substantiallyfrom that found for in vitro spindle assembly in meiotic ormitotic extracts prepared from Xenopus oocytes. Following theinitial studies of Lohka and Maller (1985), Sawin andMitchison (1991) described both sperm-spindle andinterphase-mitotic spindle assembly in Xenopus egg lysates indetail. Incubation of demembranated sperm in ‘meiotic’extracts, resulted in the formation of bipolar and multipolarsperm-spindles. Sperm-spindle assembly was the result offormation of sperm half-spindles, each composed of spermchromatin and a single sperm centrosome, which subsequentlyfused to form bipolar spindles. Alternatively, whendemembranated sperm were first introduced into ‘interphase’egg extracts, followed by the addition of mitotic lysate to drivethe system into mitosis, the bipolar spindles that formed(interphase-mitotic spindles) did not contain asters. Sincesperm-spindles, but not interphase-mitotic spindles, assembledin the absence of kinetochores, the authors proposed thatchromatin may stabilize microtubules independently ofkinetochores, and that such interactions may contribute to theprocess of spindle assembly in Xenopus oocytes and lysates(Sawin and Mitchison, 1991).

Recent studies by Heald et al. (1996) have demonstrated thatincubation of artificial DNA-coated beads in interphaseextracts leads to the assembly of chromatin (bead-chromatin)and formation of functional nuclei. When these extracts aredriven into mitosis, the bead-chromatin directs the assembly ofbipolar, anastral spindles (bead-spindles) in the absence ofeither centrosomes or centromeric-DNA sequences. Thus,neither MTOCs nor kinetochores are necessary for bipolaranastral spindle assembly in Xenopus egg extracts (Heald et al.,1996, 1997). Further, anastral spindles are known to assemblein plant cells (Bajer and Mole-Bajer, 1982), and Drosophila(Theurkauf and Hawley, 1992; McKim and Hawley, 1995;Matheis et al., 1996).

While meiotic spindle assembly can occur in the absence ofcentrosomes and asters in some animal cells, meiosis I spindleassembly in Spisula oocytes appears to involve a significantlydifferent mechanism(s). First, meiosis I spindle assemblyoccurs in the presence of centrosomes and asters, and thusrepresents a true astral spindle assembly system. Much like thesequence of events typical of mitosis in somatic animal cells,Spisula meiosis I spindle assembly involves the emergence oftwo asters which associate with a nuclear surface, followed bythe breakdown of the nucleus (GV) and the association ofchromosomes with astral microtubules. Thus, Spisula meiosisI spindle assembly is best explained by the ‘search and capture’mechanism (Kirschner and Mitchison, 1986), where dynamicmicrotubules emanating from two replicated centrosomescapture the chromosomal kinetochores after nuclear envelope

R. E. Palazzo and others

Fig. 5. Immunoblot analysis of Spisula oocyte extract using thedynein IC specific monoclonal antibody m74-1. (A) SDS-PAGE andimmunoblot of Spisula oocytes; lane 1, Coomassie-stained gel; lane2, immunoblot with m74-1. (B) Immuno-precipitation of m74-1specific antigen from Spisula oocytes. Lanes S and 1-3, Coomassiestaining of starting material (S) and immuno-precipitated fractionsusing Protein A-Sepharose beads (lane 1), m150-1/Protein A-Sepharose beads (lane 2), and m74-1/Protein A-Sepharose beads(lane 3). Lanes 1′-3′, immunostaining of immuno-precipitatedfractions using monoclonal antibody m74-1. Asterisk in lane 3indicates the position of the dynein heavy chain.

kDa

200

97

66

45

1299Dynein in spindle assembly in Spisula solidissima oocytes

breakdown, ultimately leading to the assembly of bipolarspindles. In contrast, anastral spindle assembly, which is foundin some specialized animal cells, particularly oocytes(Theurkauf and Hawley, 1992; McKim and Hawley, 1995;Matheis et al., 1996), and in plant cells (Bajer and Mole-Bajer,1982), appears to involve a ‘self-assembly’ mechanism whichis characterized by the chromatin-dependent organization ofmicrotubules to form a fusiform spindle in the absence ofMTOCs (Heald et al., 1996, 1997). No chromatin-microtubuleinteractions were detected in Spisula lysates other than theassociation of chromosomes with astral microtubules.Importantly, in the presence of either vanadate or an antibodythat is known to inhibit dynein function, the in vitro assemblyof Spisula spindles was inhibited. However, no microtubuleswere found associated with chromosomes in the presence of

this antibody. These results are in contrast to those reported forspindle assembly in Xenopus egg extracts where dyneinantibody disrupted the formation of spindle-poles anddissociated sperm asters from sperm-spindles (Heald et al.,1997), but did not inhibit the assembly of chromatin-microtubule complexes or chromatin-dependent sorting ofmicrotubules into antiparallel arrays (Heald et al., 1996). Thus,at this point, we have no evidence that chromatin-directedmicrotubule organization plays a significant role in meiosis Ispindle assembly in Spisula oocytes.

It has been suggested that different cell types may use oneor the other of these two basic mechanisms or may employaspects of both mechanisms for spindle assembly (Rieder,1991). Thus, the molecular mechanisms involved in chromatin-directed spindle assembly may not be applicable to systems

Fig. 6. Inhibition of spindle formation by dynein ICspecific antibody m74-1 analyzed by DIC (A,E)fluorescence microscopy (A′, B-D, E′, F,G).Activated extract and unactivated lysate wassupplemented with 0.2 mg/ml rhodamine-labeledtubulin and 0.5 mg/ml control antibody (A-D) orwith 0.5 mg/ml dynein IC antibody m74-1 (E-G)prior to spindle formation (see Materials andMethods). (A′ and E′) Fluorescent images ofchromosomes found in the same field as the spindleand asters shown in A and E, respectively. Bipolar(A and D) and monopolar (B and C) spindles whichcontain clusters of chromosomes (A′, B-D)associated with asters formed in the presence of thecontrol antibody. In the presence of the dynein ICspecific antibody m74-1, no monopolar, bipolar, ormultipolar spindles formed (E-G) and nochromosome clusters (E′, F and G) or aster-chromosome complexes were observed. However,m74-1 antibody had no effect on microtubule asterformation (E-G). Bar, 20 µm.

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which rely on centrosome-directed spindle assemblymechanisms, such as Spisula oocytes or somatic mammaliancells.

Aster formationHistorically, the centrosome has been the center of attention asthe major microtubule organizing center of animal cells. At theturn of the century, cytologists recognized that embryos andembryonic cell fragments could be induced to form multipleastral arrays, referred to as cytasters (for review see Wilson,1925). Further, it has been demonstrated that a surgical cellfragment from a lower vertebrate pigment cell can organize anastral microtubule array in the absence of a bona fidecentrosome (Rodionov and Borisy, 1997). Thus a long historyof research has repeatedly indicated that the cell cytoplasm hasthe capacity to organize microtubules into astral arrays in acentrosome-independent manner. Indeed, we have reportedthat addition of the microtubule polymerizing agent hexyleneglycol to cytoplasmic extracts prepared from activated Spisulaoocytes not only augments the birefringence of asters whichcontain bona fide centrosomes, but also induces the formationof secondary small asters which do not contain centrosomes(Palazzo et al., 1988). Acentrosomal aster formation has alsobeen induced in mammalian cell (Gaglio et al., 1996) andXenopus egg (Verde et al., 1991; Heald et al., 1997) extractsby treating these with the microtubule polymerizing agentstaxol or dimethyl sulfoxide (DMSO), respectively. Importantly,dynein function appears to be required for the spontaneousassembly (Verde et al., 1991) and maintenance (Heald et al.,1997) of DMSO asters in Xenopus egg extracts, and for Taxolinduced aster formation in mammalian cell extracts (Gaglio etal., 1996). Thus, a strong body of evidence suggests thatacentrosomal aster formation requires dynein. However, therequirements for centrosome assembly and centrosome-dependent aster formation are not well understood (Compton,1998).

The studies described here show that even though vanadateand dynein antibody can inhibit Spisula spindle assembly invitro, at the concentrations used they had no apparent affect onaster formation. Since these Spisula asters assembled in theabsence of microtubule stabilizing agents, and are rather largein diameter (>10 µm), they probably contain centrosomes, andare the result of centrosome assembly and maturation processesknown to occur in these lysate-extract mixtures (Palazzo et al.,1992). Taken together, these results suggest that centrosome-dependent aster formation in Spisula lysate mixtures does notrequire dynein function. Consistent with these results are thereports that although dynein antibody dissociates sperm-astersfrom sperm-spindles assembled in Xenopus lysates, the sperm-asters persist (Heald et al., 1997). Similarly, injection of dyneinantibody into metaphase mammalian cells disrupts bothspindle integrity and the association of centrosome-asters withthe central spindle, however centrosome-aster integrity remainsintact (Gaglio et al., 1997). Thus, the mechanism(s) thatgoverns centrosome-dependent aster formation appears to bedistinct from that which controls centrosome-independent asterformation, which can be artificially induced by treatment withmicrotubule stabilizing agents. Most importantly, the resultspresented here suggest that dynein function is not required forcentrosome assembly, centrosome maturation, or centrosome-dependent microtubule nucleation.

Spindle assembly under defined conditionsThe results presented indicate that aster-chromosomecomplexes, reflective of meiosis I spindle assembly, can beassembled in cell-free extracts prepared from Spisula oocytes.Determination of which molecules are necessary for spindleassembly, and elucidation of the precise function of each, willultimately require the ability to assemble spindles underdefined conditions. For Spisula oocytes, methods for thefractionation and long term storage of the key organelles andsome of the proteins required for meiosis I spindle assemblyhave already been described, and certain key cell cycle-dependent events have already been reconstituted in definedmedia. Thus, GVs and associated chromosomes can be isolated(Dessev et al., 1989) and conditions for the in vitrophosphorylation of lamin proteins which leads to GVBD underdefined conditions have already been reported (Dessev et al.,1991). Further, methods are available for the preparation ofmicrotubule proteins from marine eggs (Suprenant et al.,1991), including Spisula oocytes (Palazzo et al., 1988;Suprenant et al., 1991). Finally, centrosomes (Vogel et al.,1997; Palazzo and Vogel, 1999) and asters (Palazzo et al.,1988) can be isolated from activated oocytes in significantquantities, and isolated Spisula centrosomes retain the abilityto nucleate microtubules and assemble asters under definedconditions. Thus, two of the key events required for spindleassembly, GVBD and centrosome-dependent aster formation,have already been executed under defined conditions.Conceivably then, the reconstitution of isolated GVs,centrosomes, and tubulin in defined media, under conditionsthat would allow the simultaneous induction of GVBD (Dessevet al., 1991) and centrosome-dependent aster formation (Vogelet al., 1997) could result in the formation of spindles similarto those assembled in Spisula oocyte lysate-extract mixtures asdescribed in this report.

The authors thank Dr George Langford (Dartmouth College) for hisgenerosity in allowing the use of his video-microscopy system for theanalysis of some of the experiments described. This study wassupported in part by the National Institutes of Health (GM43264) andthe American Cancer Society (JFRA-314) to R.E.P; grant P12868-GEN from the FWF to W.S.; a Human Frontier Science ProgramGrant to S.A.K.; grant We790 from Deutsche Forschungs-Gemeinschaft to D.G.W.; the Robert Day Allen Fellowship (to R.E.P.and E.V.). and the Herbert W. Rand and MBL Fellowships (to W.S.)of the Marine Biological Laboratory.

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