A mutation (dosach) in Drosophila which affects aster formation and nuclear migration during cleavage

Biol Cell (1996) 87,45-54 0 Elsevier. Paris

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Original article

A mutation (dosach) in Drosophila which affects aster formation and nuclear migration during cleavage Sarah S Craig, Neil G Brink School of Biological Sciences, Flinders University of South Australia, Bedford Park, South Australia, Australia, 5042 (Received17February1996;accepted 3 July 1996) Summary - Herewe describea new mutant,dosuch (dos), in Drosophila melanogaster. In the mutant,centrosomes divide andinitiate spindleformation similar to that seenin wild-type embryos.Nevertheless,mutant embryosform cleavagespindlesthat lack visible astersand display abnormalmorphology, including mono- and tri-polar spindles,spindlechainsand incorrect alignment.Irregular nuclearmigrationis alsoobservedin mutantembryos,andthis may suggestthat astralmicrotubulesare importantfor spindlespacing during cleavageand alsoin maintainingtbe integrity of the mltotic apparatus.Confocal microscopyhasbeenusedto correlateorganization of microtubules,centrosomalproteinsandchromosomes in wild-type anddosach (dos) embryos. embryonic

cleavage

/ asters / microtubules

/ spindle

Introduction The most conspicuousmanifestation of the onset of mitosis is the disassemblyof the interphasic microtubule array and formation of the bipolar mitotic spindle. Microtubules, ubiquitous cytoskeletal filaments constructed principally from heterodimeric a- and P-tubulin proteins [35], are nucleated and organized in animal cells by the centrosome at all stagesof the cell cycle [31, 321. Centrosomal alterations during mitosis include changesin the phosphorylation status of proteins and increased microtubule nucleation capacity [3 11. FTubulin, a member of the tubulin superfamily, is necessary for microtubule nucleation in vivo [24, 28,

431. Such changes in cytoskeletal function are believed to be regulated by a core mitotic oscillator (p34cdc2 and cyclins) that has been evolutionarily conserved [41]. In syncytial embryos such as Drosophila the process of nuclear division (mitosis) is integrated with that of nuclear migration [ 13, 291. A number of studies have demonstrated a requirement for maternally supplied cytoskeletal components [13, 20, 691 and centrosomes [60, 681 in maintenance of nuclear spacing and migration during the syncytial cleavage divisions in Drosophila. The first 13 cleavage divisions in Drosophila occur in a syncytium, that is, nuclear division in the absence of cytokinesis [12, 44, 691. Approximately 350 nuclei migrate synchronously from the interior of the embryo to the cortex during nuclear cycles eight and nine.

DrosophiZa maternal effect mutant, (dosach), which lacks asters and astral microtubles, that has been used to investigate the role of these structures during mitosis. The results of this study suggestthat the non-kinetochore microtubules are important in establishment and/or maintenance of the

integrity of the bipolar mitotic spindle, and also in orientation of the spindle structure during mitosis, Materials and methods Genetic stocks

The originaldosallele,fs(3)7-Z67,wasisolatedfrom a collection of maternallethalsprovided by Prof R Saint (Moretti, unpublished).A screenfor other allelesgeneratedby P-elementmobilization [51] produced a secondallele, (P58), which has been usedin the studiesreportedhere. Both maternallethals,which areweakly expressed,aremaintainedin balancedstocksover the inversion‘I’M3eSbSer[34]. Deletion mappinghas localizeddos to 85D12 to SSEl-3 on the polytene chromosomemap (Craig, unpublished).The deficienciesusedin mapping,andthe regions each delete, were: Df(3R)byiO (85D8-12:E7-Fl), Df(3R)by6* (85Dll-14:F16), Df(3R)by416 (85DlO-12:El-3) and Df(3R) GBlw (85D12-ElO).The mitotic mutantabnormal anuphuse resolution, which mapsto 85E7-F16,wasfound to complementdos, establishingthat dos anduur1 are separategenes. Fination and staining of embyros

Each nucleus and its associated cytoplasm remains spatially distinct from its neighbours and maintains individual actin, intermediate filament, and microtubule arrays [29,63,65].

Fertilized eggslaid by transheterozygousdos/GB’m were used for immunohistochemicalanalysis.Eggs were laid over a 2-h

During mitosis in Drosophila prominent asters arise from the centrosome. Astral microtubules that project outwards from the aster gradually dissipate during metaphase and re-appear during early anaphase[30, 641. The roles of astersand astral (cytoplasmic) microtubules in spindle positioning and nuclear migration during the syncytial nuclear divisions are not clearly understood. Here we describe a

solution of heptane/90% methanol in PBS containing 1 mM EGTA, and shaken continuously for 10 min at room temperature. The heptane phase was removed and embryos that had sunk into the methanol phase were transferred to fresh methanol in PBS

period onto yeastedagar platesat 25°C dechorionatedin halfstrengthcommercialbleachfor 2 mm, rinsedextensively in 0.1% Nonidet, then fixed usingthe techniqueof Mitchison and Sedat [39], except that after rinsing the embryoswere placedin a 1:l

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containing 1 mM EGTA and 1 mM GTP. GTP is known to stabilize microtubules [3]. Embryos were left in the fixative for a minimum of 24 h prior to staining. Methanol fixed embryos were rehydrated as described in [60] and pre-incubated in PBS containing 1% Triton X-100 and 1% BSA over 2 h (this solution was used for all antibody incubations and rinses). Primary and secondary antibody incubations were for 18 h at 4”C, with each incubation followed by four 30-min rinses. Stained embryos were mounted in 50% glycerol in Tris (pH 7.5), containing n-propyl gallate to reduce photobleaching. Slides were examined using a Bio-Rad MRC 1000 confocal imaging system. 1 pm thick optical slices were generated and merged using software provided with the instrument. A l/200 dilution of the anti-tubulin monoclonal antibody YL1/2 (Seralab) was used to immunolabel a-tubulin. Labelling was detected using a l/200 dilution of a secondary antibody, biotinylated anti-rat IgG (Amersham), and l/200 dilution of a fluorochrome, Texas-red streptavidin (Amersham). DNA was stained with a l/25 dilution of a 1 mg/rnl chromomycin A, (Sigma) in PBS + 10 mM Mg2+ stock solution. Chromomycin preferentially binds to GC-rich DNA sequences [lo]. Centrosomes were immunolabelled with a l/500 dilution of dmapl90 or l/500 dilution of Dr2 and visualized with anti-rabbit IgG-Cy5 (Jackson Immunoresearch). Analysis of spindle dimensions Spindle length and width Spindle dimensions of 40 mutant and 18 wild-type embryos were measured using the length/profile software supplied with the confocal imaging system, and analysed using a commercial statistics program. Length measures were taken from each halfspindle of a bipolar metaphase spindle. Each half-spindle was measured from the apex to the base of the structure. For each complete spindle the separate measures were summed. Width was taken across the bipolar spindle at its widest point. Bipolar metaphase spindles only were used for this analysis. Spindle spacing The distance between neighbouring spindles was measured using the length/profile software supplied and analysed using a commercial statistics program (described above). Distance was measured from apex to apex of neighbouring spindles, with the shortest measure recorded and used in the analysis. For each spindle there were two measurements (one for each apex), except for one tripolar spindle (three points were measured). A total of 20 wildtype spindles and 91 dos/GB’@’ spindles were measured for this analysis.

Results and discussion

Microtubule behaviour during mitosis Figure 1 shows examples of wild-type and dos spindle organization at similar stages of mitosis. Wild-type spindles, organized into a bipolar structure, have well developed asters visible at each end of the spindle (fig lA, B, C, D). Astral microtubules extend out from the aster into the cytoplasm during early metaphase (fig 1A) and anaphase (fig lC, D), but are not present at late metaphase(fig 1B). Mutant spindles can be organized into a bipolar structure

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(fig 1E) but abnormal structures such as tri-polar spindles are also found (fig lF, G). Asters and astral microtubules are not present in any dos embryo at any stage of mitosis (fig lE, F, G). In mutant spindles at anaphase(for example fig 2F, open arrow) no astral microtubules are apparent, unlike wild-type anaphasespindlesin which astral microtubules re-appear (fig lC, D). Furthermore, there are variations in spindle shape and spacing during the syncytial divisions. These are shown in figure 2. Wild-type spindlesin late metaphase(fig 2B) are regularly spaced,aligned parallel to the embryo surface and uniform in appearance.The spindlesare triangular in shape and the apex is clearly defined. Each wild-type spindle is also surroundedby a lighter staining region, which presumably represents pools of a-tubulin, and corresponds to observations made by Kellogg et al [30]. In dos embryos, spindles in late metaphaseare dumb-bell shaped (fig 2H). The pools of a-tubulin are absentin theseembryos (fig 2E, H). Defects in alignment of spindles are also apparent, in the mutant as somemutant spindlescan be found positioned perpendicular to the surface of the embryo (fig 21, closed arrow). No significant differences between the meansfor spindle length or width were found (data not shown). Overall, metaphase spindle dimensions thus appear similar in both mutant and wild-type embryos. This finding is not surprising, since abnormal spindles(such astripolars, monopolars, chains) were excluded from the mutant group. However, significant differences were found between the variances for both length and width (data not shown). This suggests that differences between individual wild-type and mutant spindles are not due to sample variation, but may reflect fundamental differences in microtubule structure and function. Asynchrony is also apparent in mutant embryos with metaphase (fig 2F, closed arrow) and anaphase(fig 2F, open arrow) figures clearly evident within the same embryo, in contrast to wild-type embryos in which nuclear division is synchronous(fig 2B). Although bipolar spindles are present, mis-alignment of opposing half-spindles is clearly seenin a dos embryo during anaphaseB (fig 3C). None of the spindlespresent have astersor astral microtubules. Spindle pole separationin this embryo appearsunperturbed, and resemblesthat of wildtype embryos (data not shown). There is evidence that astral microtubules may be involved in the early stagesof formation and stabilization of a bipolar spindle [61], and in spindle pole separationduring anaphaseB [ 1, 231. The forces acting to establishand maintain metaphase spindle alignment are mediated partly by parallel interdigitating cytoplasmic (astral) microtubules, and also require the action of microtubule motor proteins (reviewed in [37]). The functions of kinesin-related proteins in mitosis are reported to include MTOC migration in prometaphaseand maintenance of bipolarity (bimC group), poleward movement of chromosomesand spindle microtubules (ncd and KAR3), and anaphaseB spindle elongation (CHOl) (see

Fig 1. Localization of a-tubulin in 0-2-h-old wild-type (A-D) and dos/GB lo4 (E-G) embryos, examined by confocal microscopy. Embryos were fixed and stained as described in Materials and methods. Wild-type embryos arrows show: early metaphase with prominent aster and well defined astral microtubules (A); aster in late metaphase, with absent astral microtubules (B); asters and reforming astral microtubules in early anaphase (C, D). Mutant dos embryos: probable metaphase spindle that lacks asters and astral microtubules (E); fused tri-polar (upper) and bi-polar (lower) spindles, probable late metaphase with no asters or astral microtubules (F); probable anaphase &i-polar spindle that lacks asters and astral microtubules (G).

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[6], and references cited therein). Cytoplasmic dynein, a minus end directed motor protein localized to the mitotic spindle and kinetochores at the onset of mitosis [46,59,62], may be involved in maintenance of intercentrosomal distance prior to prometaphase [61], as the injection of antibody to cytoplasmic dynein leads to the inward collapse of the separated spindle poles. In the fungus Nectria haematococca, ablation of portions of the central spindle region increases the rate of spindle pole separation, and damage to one aster promotes the migration of the entire spindle in the direction of the opposing astral forces: spindle morphology in the absence of astral microtubules is also abnormal [l]. In contrast, dos embryos generally can form bipolar spindles and undergo spindle pole separation in the absence of asters and astral microtubules. MTOC migration, bipolarity and spindle pole separation are essentially normal in comparison to wildtype embryos. It is possible that the dynamic behaviour of microtubules in mitosis reflects a balance between many components, complicating analysis of a single gene product. The subtle differences between wild-type and dos embryos in spindle morphology, intercentrosomal distance, and orientation may reflect a degree of functional redundancy [19] for the dos gene product. For example, in Saccharomyces cerevisiae, Cin8p and Kiplp act redundantly in mitosis to separate spindle poles and prevent inward collapse of the separated poles [25,52,53]. The second feature seen in mutant dos embryos is random asynchrony during the syncytial divisions. Progression through nuclear division cycles l-9 is synchronous in Drosophila [57] and the later syncytial blastoderm divisions are propagated as a pole to equator wave-front, ie ‘metachronous’, in which nuclei in a common front are at the same stage of mitosis [13]. It is not fully understood how synchrony is generated or maintained, but interactions between components of the mitotic oscillator and spindle microtubules may contribute to a spindle feedback mechanism. Degradation of cyclin B, which closely associates with the polar regions of the spindle and spindle microtubules in a cell-cycle dependent manner [48, 361, precedes the metaphase to anaphase transition. Inhibition of cyclin B degradation leads to metaphase arrest, either as a consequence of interfering with the cyclin degradation pathway directly, for examplefizzy mutants [9] or indirectly by interference with microtubule dynamics, for example microtubule inhibitor studies [67]. Alternatively, spindle feedback may be generated through the interaction of chromosomes and spindle fibres [38], with anaphase delayed until all chromosomes are correctly positioned on the metaphase plate. The metaphase organization of chromosomes in dos embryos differs from wild-type embryos and lagging strands are occassionally seen.

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Centrosome behaviour during mitosis

Centrosomes have previously been shown to be involved in aster formation [ 11, 24, 42, 581. Therefore, in order to assess the role of centrosomes in spindle assembly we stained embryos with antibodies (dmapl90 or Dr2) to Drosophila ytubulin. Our results are shown in figure 3. Anti-Dr2 antibody is associated with the spindle poles in both wild-type (fig 3A) and dos embryos (fig 3B). Co-localization of ‘y- and a-tubulin antibodies in both wild-type (fig 3A) and mutant embryos (fig 3B) was found to be similar. We found that dmapl90 in wild-type embryos is associated with the centrosome and also faintly stains spindle microtubules during mitosis (data not shown), a distribution similar to that found by Raff et al [48]. This was also found to be the case in dos embryos where anti-dmapl90 antibody is associated with the spindle poles, in the region corresponding to the centrosome (fig 3C) and also stains microtubules in the spindle region (data not shown). The centrosome cycle in Drosophila can be uncoupled from the nuclear cycle in wild-type embryos injected with aphidicolin [47]. Centrosomes in these embryos can complete migration to the cortex and induce normal cytoskeletal re-arrangements, such as actin capping and pseudo-cleavage furrow formation. Mutations in Drosophila have been isolated that uncouple the centrosome cycle from other cell cycle events, for example giant nuclei [14, 151, plutonium and pan gu [55], aurora, merry-go-round and polo [18]. Unlike these mutants, uncoupling of centrosomes from spindle formation is not a feature of dos embryos, and the distribution of dmapl90 and ytubulin is similar to that in wild-type embryos during metaphase and anaphase. Although the centrosome cycle appears normal in the dos embryos, astral microtubules are clearly not initiated and/or stabilized. Hence, we cannot exclude the possibility that centrosomal protein(s) involved in centrosome maturation or microtubule dynamics may be defective. This caveat also applies to dmapl90 and ytubulin that may be present in the embryo but are functionally impaired. Chromosome congression and segregation

Since the principal function of the mitotic spindle is segregation of chromosomes we were interested in the effect dos embryos, which display abnormal spindle structure, would have upon this process. We have observed differences between wild-type and dos embryos at similar stages of mitosis (figs 2, 3). Figure 2 shows syncytial blastoderm wild-type and dos embryos at metaphase. The chromosomes of individual nuclei in the mutant are ovoid in appearance (fig 2G), and splay out from the metaphase plate (fig 21) in contrast to the appearance of metaphase wild-type chromosomes (fig 2A)

4 Fig 2. Double labelling of wild-type (A-C) and dos/GB 104(D-I) 0-2-h-old embryos.Embryoswere fixed and stainedasdescribedin Materials and methods, andexaminedby confocal microscopy.A, D, G. DNA (chromomycinA3). B, E, I-I. Microtubules(a-tubulin). C, F, I. Merged imageof DNA (green)and cc-tubulin(red). Wild-type embryoat metaphase. A. DNA regular,compactarrangementof chromosomes, all in focal plane.B. a-Tubulin showingregular array of spindlesin samefocal plane, similarsize and shape.Stained regionsaroundindividual spindlesprobablyrepresentpoolsof a-tubulin. C. Merged imageof DNA (green)and a-tubulin (red). Mutant embryosDNA (D, G); a-tubulin (E, II); mergedimageof DNA (green)anda-tubulin (red) (F, I). Arrows showexamplesof differences betweenmutantandwild-type embryos:(D, G, I) metaphase configurationof chromosomes not ascompact(openarrow). D. Anaphase configurationwith laggingstrand(closedarrow).F. Metaphase(closedarrow) andanaphase (openarrow) in this mergedimageof DNA andspindlesclearly showasynchronyin the mutant. Asynchrony is alsoseenin D with metaphase (openarrow) andanaphase (closed arrow) figurespresent.I. M&orientation of spindle(closedarrow) out of the focal plane. Abnormal mutantspindlesdiffer from wildtype asseenin E andI-I, openarrow); theseembryosalsolack poolsof a-tubulin.

b t. r :, ;: ;, --

Fq 4. Spindle distribution in syncytial blastoderm $d-type (A) and dos/GBlo4 (C, D) embryos, and cycle 3 (B) dos/GBJo4 embryo. Embryos fixed and stained as described in Materials and methods and exsmined using confocal microscopy. Syncytial blast@derm embryos: wild-type spindles (A) show regular arrangement, all in focal plane and regions of rz-tubulin surrounding each spindle. Spindles are of uniform appearance. Mutant spindles (C, D) show irregular ~&tribution, abnormal morphology, and lack pools of a-tub&n around each spindle: Left arrow CD) shows: chains of linked spindles and right arrow (D) shows a c- fused tripolar spindle. A pre-migratory cycle 3 &s/G&J4 embryo shows that abnormal spindle mor‘~,phology manifests early (B). Arrow points to a narrow monopolar spindle. b

;~ Fig 3. Double labelled O-2-h-old wild-rype (A) Andy i dqs/GW4 embryos (B) at prophase, stained for CXtubulin (red) and ytubulin (green, or yellow where f overlap-with a-tubulin occurs). FTubulin distribution , in. both cases appears similar. Triple labelled 0-2-h. old dos/GB’04 embryo (C) at anaphase B. D#A H. (green, or yeIlow where overlap with a-tubulin $: -@curs), a-tubulin (red) and ytubulin (blue). y’l’ubui lin distribution is similar to wifd-type embryos (no1 1 shown) associating with spindle poles. It is also ass+ L. ciated with spindle microtubules (not shotin). Also 5 note chromosome segregation appears unperh&e& @ The spindles shown, whilst bipolar, lack asters and ii: astral microtubules and epposing half-spindles are in i some case-s misaligned. Embryos fixed and stained i as described in Materials- and methods, and extined using confocal microscopy.

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SS Craig,

on the metaphase plate (fig 2C). Chromosome alignment on the metaphase plate is less compact in dos embryos (fig 21) in comparison to that of wild-type chromosomes (fig 2C). Although lagging strands are occasionally found in the mutant (fig 2D, closed arrow), anaphase segregation in general was found to occur normally (fig 3C) when compared to wild-type embryos (data not shown). Lagging strands are observed in conjunction with abnormal spindles that are mis-aligned or mis-shapen. Mutant spindles (fig 2E, H), whilst bipolar, are clearly different from wild-type spindles (fig 2B). Nuclear behaviour and distribution in wild-type Drosophilu embryos has previously been described [4, 121. Earlier studies in monoastral sea urchin embryos found that astral, but not spindle, microtubules rapidly depolymerize in response to nocodazole [27]. In these embryos congression and segregation of chromosomes still takes place, despite abnormal alignment of chromosomes on the metaphase plate. This suggests that astral microtubules, while not essential for chromosome congression or segregation, may have a role in the metaphase alignment of chromosomes. .In dos embryos that form bipolar spindles the effects upon chromosome alignment are subtle, with segregation able to occur normally. This suggests that the kinetochore or motor proteins involved in chromosome congression/disjunction are able to function in the mutant (see [16] for review) and that astral microtubules are not essential for these processes. Spatial distribution divisions

of spindles during syncytial cleavuge

We wished to evaluate whether abnormal spindle structures had any effect on nuclear migration during cleavage. Figure 4 shows spindle distribution in syncytial blastoderm wild type and dos embryos (in which nuclei have completed migration to the cortex) and a dos embyro during cycle 3 (where nuclei are located in the interior of the embryo). Wild-type embryos in metaphase (fig 4A) have a regular distribution of spindles on the surface and all are in the plane of focus. In dos embryos (fig 4B, C, D) abnormalities in spindle morphology and distribution are variable, ranging from relatively normal to grossly disturbed. Although the number of spindles in cycle 3 mutant embryos (fig 4B). is normal, the morphology is abnormal. In this dos embryo opposing half-spindles, in those that are bipolar, can be seen to be n&-aligned. Some spindles have a narrow half-spindle with a focal point of a-tub&n staining opposite (arrow, also upper right). This point represents tubulin that has not been organized into a half-spindle. Abnormal spindles are thus apparent from a very early stage in dos embryos. Spindles in post-migratory dos embryos of similar age (fig 4C, D) are unevenly distributed, with variations in shape and arrangements. Shape varies from bipolar bat dumbell shaped (fig 4C) to grossly abnormal arrangements (fig 4D) including chains (arrow, left), mono-polar, &i-polar (arrow, right). Mutants also lack the region of lighter staining seen in wild-type embryos. The significance of absent a-tubulin staining around individual spindles in the mutant, which is present in wild-type embryos (fig 4A) is unclear, but perhaps may contribute to a pool of sub-units that could be utilized in microtubule polymerization. Mean distance between neighbouring spindles (described in Materials and methods) is significantly different in mutant embryos (table I). Wild-type spindles tend to be distributed at regular intervals over the surface of the embryo (range 5.81 pm) in contrast to mutant embryos (range 13.98 pm). There is also a significant difference between the variances

NC;

Brmk

I. Distance (pm) between neighbouring spindies m spncytial blastoderm embryos. 1------.- -.... Wild-type dos/GB lo4

Table

.___----_-~ II

Meana SD

Varianceb

II_-

40 6.37 pm 1.59 pm 2.54 pm

183 5.17 pm 2.37 j.lm 5.63 grn

“f-test (assuming unequal variances) t 3.71, P (T < E) two-M 3.75 x lW, Tcriticai1.99. bF-test (two sample for variances) F 2.22, P (F i f) one-tail 2.14 x 10-3, Fcritica,0168.

(table I). This suggests that wild-type embryos are able to organize their spatial ~arientation utilizing a mechanism that is absent or impaired in dosaeh embryos. The reduced- numbers of spindles in post-migratory mutant embryos may reflect a failure in migration, since there is evidence implicating astral microtubules in this process [4,20,29]. In Drosophila equidistant spacing of wild-type. nuclei is dependent upon intact astral microtubules [20, 691. Disrup; tion of asters and astral microtubules interferes with the processes of nuclear migration and spindle positioning within the syncytial Drosophila embryo. A role for other filament systems (for example actin) and the centrosome in these processes also has been demonstrated [4,20,29,69]. Nuclear migration. in early cleavage wild-type Drosophila embryos is disrupted following cytochalasin treatment, and mirrored in the nuclear migration behaviour of the mutant N441 [20]. The partitioning defective (par) genes par-2 and par-3 affect centrosome m&raEiOn and subsequently spindle orientation in Caenorhabditis elegans during then first and second cleavage divisions [8]; these events are sensitive to cytochalasin pulses in the first division cycle [22]. Pelvetia fastgiata embryos treaied with drugs that disrupt microfilament (cytochalasin. D) and microtubule (nocodaiole) organization also display dis rupted~nuclear roEation [2]. In Saccharomycses cerevisiae both a dynein-like protein DYNl [lo, 33) and .act5, an actin related protein- [4@ are required for normal spindle positoning and nuclear migra:tion; these processes are also interfered with in yeast p. tubulin mutants [451 and Sepl mutants 1261. In yeast, dynein function requires .dynactin complex [ 17, 541. -Vertebrate Arpl (which forms part of the dynactin complex) shows homology to act5 [40]. In Neuruspuru crassa Ehe-gene rri$. identified as an actin-related protein with similarity to centromere associated actin-RPV/centractin, also affects nuclear migration [SO]. Actin-RPV/centractin is involved in linking actin filaments to cytoplasm& microtubules [21].It has been suggested that cytoplasmic (astral) microtubules interact with defined cortical sites (such as the actin cytoskeleton) to influence spindle orientation and nuclear migration 145, 66). Defects in proteins (for example cytoplasmic dynein) that mediate such interactions would lead to abnormalities in spindle position and migration, which both occur in dos embryos. Thus the general spindle disorganization and abnormal nuclear migration present in dos embryos could be attri.buted to defects in nucleation or stabilization--of astral microtubules. Consequently, this may lead to failure to mediate interactions between astral microtubules, and other cytoskeletal or centrosomal components present in the early

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embryo. The gene is currently being cloned to determine the nature of the gene product and how these apparently diverse phenotypes are integrated to a common cause. Identification of such gene products will further our understandingof the cell cycle, a fundamental but complex process.

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Acknowledgments

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The authors would like to thank Prof DM Glover for providing aart, Dr Peter Kolesik for assistance with confocal microscopy work undertaken at the Waite Institute, and Dr BM Alberts and Dr Y Zheng for providing dmapl90 and Dr2 antibodies, respectively.

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