Research Article 3797 RanGTP is required for meiotic spindle organization and the initiation of embryonic development in Drosophila

J. Cesario and K. S. McKim* Waksman Institute and Department of Genetics, Rutgers, the State University of New Jersey, 190 Frelinghuysen RD, Piscataway NJ 08854-8020, USA *Author for correspondence ([email protected])

Accepted 4 July 2011 Journal of Cell Science 124, 3797–3810 ß 2011. Published by The Company of Biologists Ltd doi: 10.1242/jcs.084855

Summary RanGTP is important for chromosome-dependent spindle assembly in Xenopus extracts. Here we report on experiments to determine the role of the Ran pathway on microtubule dynamics in Drosophila oocytes and embryos. Females expressing a dominant-negative form of Ran have fertility defects, suggesting that RanGTP is required for normal fertility. This is not, however, because of a defect in acentrosomal meiotic spindle assembly. Therefore, RanGTP does not appear to be essential or sufficient for the formation of the acentrosomal spindle. Instead, the most important function of the Ran pathway in spindle assembly appears to be in the tapering of microtubules at the spindle poles, which might be through regulation of proteins such as TACC and the HURP homolog, Mars. One consequence of this spindle organization defect is an increase in the nondisjunction of achiasmate chromosomes. However, the meiotic defects are not severe enough to cause the decreased fertility. Reductions in fertility occur because RanGTP has a role in microtubule assembly that is not directly nucleated by the chromosomes. This includes microtubules nucleated from the sperm aster, which are required for pronuclear fusion. We propose that following nuclear envelope breakdown, RanGTP is released from the nucleus and creates a cytoplasm that is activated for assembling microtubules, which is important for processes such as pronuclear fusion. Around the chromosomes, however, RanGTP might be redundant with other factors such as the chromosome passenger complex.

Key words: Meiosis, Mitosis, Microtubule, Drosophila melanogaster, Acentrosomal spindle, Chromosome segregation Journal of Cell Science Introduction extracts that lack centrosomes. Chromatin-mediated microtubule Ran is a member of the Ras family of small GTP-binding assembly depends on the presence of RanGTP in Xenopus proteins. It was originally discovered for its role in shuttling extracts (Carazo-Salas et al., 1999). Similarly, depletion of RCC1 proteins with nuclear localization sequences (NLS) into the results in a failure to form microtubule asters. Addition of nucleus (Moore and Blobel, 1993). Ran cycles from an active RanGTP to these RCC1-depleted eggs is sufficient to induce self- state, RanGTP, to an inactive state, RanGDP. The conversion of organization of microtubule asters (Ohba et al., 1999). Disruption RanGDP to RanGTP is stimulated by the chromatin bound of RanGTP levels also affects mitotic spindle assembly in guanine nucleotide exchange factor RCC1 (Bischoff and mammalian (Clarke and Zhang, 2008; Kalab et al., 2006), Ponstingl, 1991). Conversely, the conversion of RanGTP to Drosophila melanogaster (Silverman-Gavrila and Wilde, 2006) RanGDP is facilitated by the cytoplasmic GTPase-activating and Caenorhabditis elegans (Askjaer et al., 2002; Bamba et al., protein RanGAP (Bischoff et al., 1994). During G2, the nuclear 2002) cells. These results suggest that RanGTP is a major envelope creates a barrier where active RanGTP can only be contributor to spindle assembly. found within the nucleus, because RCC1 is chromatin bound. The We have undertaken an analysis of Ran function in the importin complex, which consists of importin a and importin b, Drosophila oocyte because several aspects of oogenesis and is capable of binding proteins with NLS and transporting them embryogenesis depend on microtubule dynamics (Dix and Raff, into the nucleus. Once inside the nucleus, RanGTP binds 2007; Roth and Lynch, 2009). In Drosophila oocytes, as in many importin b, releasing importin a and NLS containing proteins oocytes, meiosis is acentrosomal. Spindle assembly occurs (Clarke and Zhang, 2008). without the guidance of the microtubule organizing centers at Ran also has a role in spindle assembly by releasing spindle the poles. In this situation, the chromosomes play an important assembly factors from the repressive importin complex (Kalab role in spindle assembly. Nuclear envelope breakdown (NEB) is and Heald, 2008). The production of RanGTP near chromatin and followed by the accumulation of microtubules around the conversion to RanGDP in the cytoplasm can lead to the formation chromosomes (Matthies et al., 1996; Theurkauf and Hawley, of a gradient of active Ran that is capable of triggering 1992). The subsequent bundling and tapering of these chromosome-mediated spindle assembly (Caudron et al., 2005). microtubules by motor proteins results in a bipolar spindle. The role of RanGTP in chromosome-mediated spindle assembly Thus, Drosophila oocyte chromosomes carry a signal that has been most clearly shown by its activity in Xenopus laevis egg promotes spindle assembly when released into the cytoplasm 3798 Journal of Cell Science 124 (22)

upon NEB. However, it is unclear, what are the components of Expression of ranT24N has a dominant-negative effect this signal. To determine whether expression of the mutant forms of ran Meiosis in Drosophila arrests at the first division (Theurkauf would cause lethality similar to the loss-of-function mutant, we et al., 1993). When the oocyte then moves down the oviduct, it expressed the transgenes using P{tubP-GAL4}, which induces becomes activated and the two meiotic divisions are completed. ubiquitous expression of UAS transgenes (Lee and Luo, 1999). Independently, fertilization occurs and the sperm centriole Expression of ran+ had no effect on viability and was able to recruits microtubules that are required to bring together the rescue the lethality of a ran mutation (Table 1). Furthermore, male and female pronuclei. Finally, the nuclear membranes of the dividing neuroblasts from third instar larvae exhibited properly two nuclei fuse prior to the first mitotic division. All these events assembled spindles with no detectable abnormalities during depend on maternally contributed proteins and are thus a function metaphase and anaphase (supplementary material Fig. S1). The of the oocyte. We have examined the role of the Ran pathway in location of wild-type Ran was examined using antibodies to the these early developmental processes. We found that RanGTP has HA epitope tag that was fused at the N-terminus of the ran a role in pronuclear fusion in the embryo, is active in promoting transgenes. Ran was nuclear during interphase and then microtubule assembly in the oocyte cytoplasm, but it might not overlapped with the spindle during metaphase and anaphase be required for their recruitment of microtubules by the meiotic (supplementary material Fig. S1). Similar to the localization chromosomes. pattern in embryos (Trieselmann and Wilde, 2002), Ran was not detected on the chromosomes. Unlike ran+, ubiquitous expression of ranT24N or ranQ69L resulted in lethality of the Results embryos or at an early stage of larval development (Table 1). Generation of dominant ran mutants These results suggest that expression of ranT24N or ranQ69L has a Ran is required for mitosis (Silverman-Gavrila and Wilde, 2006) dominant effect and disrupts the normal functioning of the Ran and a mutation in the ran gene (G0075) causes lethality in pathway. Drosophila (Peter et al., 2002). Furthermore, mutations in the Drosophila RCC1 homolog, Bj1, cause lethality, and germline Maternal expression of ranT24N and ranQ69L causes sterility clones do not make oocytes (Shi and Skeath, 2004) (K.S.M., To examine the function of Ran in oogenesis and embryogenesis, unpublished results). Because these properties make it impossible the ran transgenes were expressed using the P{GAL4:VP16- to study ran mutations in oocytes, we generated mutations nos.UTR}MVD1 driver, and then genetic assays were performed predicted to be dominant alleles of ran. Previous studies in a to measure fertility and the frequency of X-chromosome variety of systems have characterized mutations that lock Ran in nondisjunction. This driver typically overexpresses UASP either the GDP (inactive) or GTP (active) states (Kahana and transgenes in oocytes (Jang et al., 2007; Van Doren et al., Cleveland, 1999; Trieselmann and Wilde, 2002). Because Ran is 1998). Differences in expression levels between different highly conserved, these same changes can be made in insertion lines were assayed by western blotting and found to Drosophila. Transgenes were made by fusing the coding region be minimal (data not shown). When the wild-type ran transgene of the wild-type ran or mutant variants to three copies of the HA was expressed, levels of fertility were normal (Table 2).

Journal of Cell Science epitope tag at the N-terminus. They were also put under the Therefore, expressing wild-type Ran does not have deleterious control of the UASP promoter, which allows for inducible effects on embryonic development. germline expression regulated by a second transgene expressing Expression of ranT24N in the female germline caused a drastic GAL4 (Rorth, 1998). To generate a GDP-locked mutant, the reduction in fertility, with an average of only 6.8 progeny per + T24N P{w ; UASP:ran } transgene was constructed with an amino female parent compared with 62.1 progeny per female expressing acid substitution of threonine to asparagine at position 24, wild-type Ran (Table 2). P{GAL4:VP16-nos.UTR}MVD1 also T24N hereafter referred to as ran . To generate a GTP-locked drives expression in the male germline. Males carrying this driver + Q69L mutant, the P{w ; UASP:ran } transgene was constructed and ranT24N were sterile, suggesting that RanGTP has an essential with an amino acid substitution of glutamine to leucine at role in male meiosis. Expression of ranQ69L in the oocyte resulted position 69, hereafter referred to as ranQ69L. For each allele, at in complete sterility (Table 2). These effects of ranT24N and least three transgenic lines were examined for expression levels ranQ69L on fertility indicate that there is an important role for and phenotypes. For each experiment, flies with these mutations RanGTP in either meiosis, fertilization or the embryonic were compared with flies expressing a wild-type transgene divisions. Therefore, we examined oocytes and embryos in (P{w+; UASP:ran+}), hereafter referred to as ran+. more detail in order to determine why the ran mutants are sterile.

Table 1. Effect of ran mutants on viability Transgene Progeny expressing Ran Progeny not expressing Ran ran+ a 755 326 ranT24N a 0 464 ranQ69L a 0 1405 ranG0075/Y; ran+ b 66 307

aEach transgene was expressed by crossing to P{tubP-GAL4}/TM6, Tb. Progeny expressing a ran transgene were Tb+. For each transgene at least two independent insertions were scored, both of which gave similar results. Stocks containing the ran mutant transgenes are viable and fertile, indicating that the lethality is specific and depends on the presence of the Gal4 driver. branG0075/FM7 females were crossed to P{w+; UASP:ran+}/+; P{tubP-GAL4}/+ males. 25% of the progeny were expected to inherit both the driver and transgene. Meiotic spindle assembly 3799

Table 2. Fertility and nondisjunction phenotypes by ran transgenes X-chromosome Transgenea Regular progeny nondisjunction progeny Progeny/female parent (n) Nondisjunction (%) ran+ 2481 1 62.1 (40) 0.1 ran+ (no driver) 1301 0 54.2 (24) 0.0 ranT24N 498 2 6.8 (74) 0.8 ranT24N (no driver) 1542 0 51.4 (30) 0.0 y w; ranT24N 619 7 14.6 (43) 4.1 Bwinscy/w 1596 0 61.4 (26) 0.0 Bwinscy/w; ranT24N 408 61 3.6 (56) 35.6 ranQ69L 0 0 0.0 (80) – ranQ69L (no driver) 2163 0 80.1 (27) 0.0 subDNT 0 0 0.0 (20) – subDNT; ran+ 0 0 0.0 (20) – subDNT; ranT24N 338 3 34.1 (10) 1.7

aEach transgene was expressed by crossing to the P{GAL4::VP16-nos.UTR}MVD1 driver and then crossed as described in the Materials and Methods. For each transgene at least two independent insertions were scored.

Two components of the Ran pathway, RCC1 and RanGAP, globular structures, a gradient of RanGTP, with the highest are present in the Drosophila oocyte concentration around the chromosomes, might not exist. The Drosophila oocyte develops in a cyst along with 15 nurse cells. Over the course of 3–5 days, the oocyte grows in size while Expression of RanT24N affects spindle pole organization in in a diplotene–diakinesis-like state, eventually receiving most of Drosophila oocytes the nurse cell components by the time they reach stages 13 to 14. To examine the effect of Ran on meiotic spindle assembly, wild- Following NEB in the oocyte, microtubules accumulate around type and mutant UASP:ran transgenes were expressed using the chromosomes, which are bundled together into a karyosome the P{GAL4:VP16-nos.UTR}MVD1 driver. Immunofluorescence (Matthies et al., 1996; Theurkauf and Hawley, 1992). This is assays of mature oocytes showed that wild-type Ran surrounds followed by the extension of poles and lengthening of the spindle. the metaphase I spindle (Fig. 2A). Interestingly, this localization Our previous work has shown that the central spindle is important pattern showed almost no overlap with the spindle microtubules, for organizing bipolarity (Jang et al., 2005), which can be unlike the pattern observed in mitotically dividing neuroblasts detected by staining for Subito, a kinesin 6 that localizes to the and embryos. Additional accumulations of Ran were found antiparallel microtubules of the central spindle. Meiosis arrests at adjacent to the clusters of RanGAP that form throughout the metaphase I until the oocyte passes down the oviduct and cytoplasm, but there was generally no overlap (Fig. 3A). In these becomes activated, at which point the two meiotic divisions oocytes expressing wild-type Ran, spindle and karyosome

Journal of Cell Science occur. morphology were normal (Fig. 2A; Table 3). A low frequency A gradient of RanGTP with the concentration highest near the of abnormal-looking spindles is expected because some of the chromosomes can be established if RCC1 is enriched on the oocytes are in early prometaphase when the spindle is first chromatin and RanGAP is in the cytoplasm. We stained mature assembling. In addition, Subito localized normally to the central oocytes with an antibody raised against RCC1 (Frasch, 1991) and spindle (supplementary material Fig. S3). Therefore, expression found that it localized around the outside of the karyosome of the HA-tagged wild-type Ran does not grossly affect spindle (Fig. 1A). By contrast, RanGAP (Kusano et al., 2001) was bipolarity or morphology. localized to globular structures throughout the oocyte cytoplasm Ran that was locked in the inactive GDP state had a different (Fig. 1B). Although the oocyte contains many vesicles, the localization pattern to wild-type Ran in mature oocytes. RanT24N localization pattern of RanGAP did not correspond to structures accumulated closely around the chromosomes (Fig. 2B), rather detected by a Lamin antibody (supplementary material Fig. S2). than around the outside of the spindle as does wild-type Ran. This These results show that RCC1 and RanGAP are located in localization is similar to the localization of RCC1 (Fig. 3B), discrete locations within the oocyte during assembly of the consistent with the RanT24N protein binding to RCC1 but not meiotic acentrosomal spindle. However, because the oocyte is being converted into the GTP form. There was some variation in large relative to the meiotic spindle and RanGAP appears in the RanT24N staining pattern; although it was always tight around

Table 3. Characterization of meiotic figures in wild-type and mutant ran oocytes Transgenea Oocytes Abnormal spindle Abnormal karyosome z-value ran+ 39 4 10% 0 0% ranT24N 16 8 50% 8 50% 2.9b ranQ69L 18 11 61% 4 22% 3.7b mars1 11 9 82% 0 0 4.9b

aEach transgene was expressed by crossing to the P{GAL4::VP16-nos.UTR}MVD1 driver. For each transgene at least two independent insertions were scored, both of which gave similar results. bP#0.01. 3800 Journal of Cell Science 124 (22)

Fig. 1. Localization of RCC1 and RanGAP in mature (stage 14) oocytes. DNA is in blue and tubulin is in green. (A) Wild-type oocyte stained with RCC1 antibody (red). (B) Wild-type oocyte stained with RanGAP antibody (red). (C) A low magnification view of the same oocyte as in B showing the clusters of RanGAP located throughout the ooplasm. The arrow points to the karyosome. Scale bars: 10 mm.

the karyosome (Fig. 3B), in some images there was staining away mutation were able to assemble a bipolar spindle but failed to T24N

Journal of Cell Science from DNA as well (Fig. 2B). Oocytes expressing ran did not properly taper the microtubules at the poles (Fig. 2E). To test appear to have a problem initiating the assembly of microtubules whether these similarities could be the result of ranT24N mutant around the chromosomes or building a bipolar spindle. However, oocytes failing to activate Mars, we stained ranT24N mutant the ranT24N-expressing oocytes had an increased frequency of oocytes with an antibody against Mars (Tan et al., 2008). In wild- abnormal spindle and karyosome organization (Table 3). The type or ran+-expressing oocytes, Mars colocalized with tubulin, microtubules were often not tapered at the spindle poles except at the spindle poles and the central spindle (Fig. 4A). By (Fig. 2B). Furthermore, the chromosomes were frequently contrast, approximately 50% of oocytes expressing ranT24N failed disorganized and failed to condense into a single round or oval to localize Mars to the meiotic spindle (Fig. 4B–D), which was karyosome (supplementary material Fig. S4). By contrast, Subito significantly different from ran+ oocytes (z53.6, P#0.01). localized correctly (supplementary material Fig. S3), suggesting Therefore, a Mars localization defect might contribute to the the central spindle was able to form in these mutants. These spindle-tapering defect observed in ranT24N mutant oocytes. results suggest that the Ran pathway has a role in organizing the These results are consistent with the conclusion that the RanGTP meiosis I spindle poles, but it might not be essential for initiating pathway is not essential for the initiation of acentrosomal spindle chromosome-based microtubule assembly or for regulating assembly in Drosophila oocytes, but might have a role in tapering central spindle proteins like Subito. the poles. Because RanGTP promotes spindle assembly through the Another spindle assembly factor regulated by the RanGTP release of spindle assembly factors, we tested whether ranT24N pathway is the microtubule-associated factor Transforming acidic mutant oocytes showed evidence of downregulating proteins coiled-coil, or TACC (Kalab and Heald, 2008) (see Discussion). known to be regulated by the Ran pathway. Mars is the TACC localizes to the poles of the meiotic (Cullen and Ohkura, Drosophila homolog of HURP, a spindle assembly factor 2001) and mitotic spindle (Giet et al., 2002) where it contributes regulated by RanGTP (Wilde, 2006) and has been shown to to the localization of Msps (Minispindles). To examine the have a role in the attachment of the centrosome to the mitotic localization of TACC during female meiosis, we expressed a spindle during Drosophila embryogenesis (Tan et al., 2008; Yang GFP fusion gene under the control of a ubiquitin promoter and Fan, 2008; Zhang et al., 2009). To study the role of Mars in (Gergely et al., 2000). As expected, in wild-type oocytes TACC meiotic spindle assembly, we examined mar1, which is a null localized to the poles of most metaphase I spindles (Fig. 4E,F). allele that deletes part of the coding region (Tan et al., 2008). There was some variation in this pattern, with TACC tending to Like ranT24N mutants, mature oocytes homozygous for the mars1 be less focused at the poles of shorter spindles. By contrast, in all Meiotic spindle assembly 3801

Fig. 2. Effect of Ran on spindle morphology in mature oocytes. The transgenes in these and all subsequent experiments were expressed using the P{GAL4::VP-nos.UTR}MVD1 driver. DNA is in blue, Ran proteins are in red and tubulin is in green. Ran was detected using an antibody to the HA tag fused to either wild-type ran (A), ranT24N (B)orranQ69L (C,D). The images in A–C are high magnification

Journal of Cell Science images centered on the karyosome. The image in D is of the same oocyte as in C but at lower magnification to show the localization of mutant RanQ69L in the oocyte cytoplasm. The arrow in D points to the karyosome. (E)Inmars1 mutant oocytes, the microtubules often fail to be properly tapered at the spindle poles. Scale bars: 10 mm.

ranT24N oocytes TACC failed to show enrichment towards the X-chromosomes that were expected to have a crossover in poles and was present next to the DNA (Fig. 4G,H). The failure greater than 95% of meioses (Baker and Hall, 1976). In these to properly localize both Mars and TACC in ranT24N mutant experiments, we found a low frequency of nondisjunction among oocytes is consistent with loss of RanGTP activity, but the the few progeny from ranT24N-expressing mothers, showing that mislocalization of spindle assembly factors is not severe enough X-chromosome segregation was not substantially affected by to prevent spindle assembly. loss of RanGTP (Table 2). These results are consistent with fluorescence in situ hybridization (FISH) experiments, which Achiasmate chromosome segregation is abnormal in showed that ranT24N and ranQ69L mutants were able to properly ranT24N mutants orient their homologous chromosomes at metaphase I To determine whether the spindle organization defects in the ran (supplementary material Fig. S5). Thus, although the spindles mutant females were associated with errors in chromosome are not properly tapered and the karyosome is disorganized in ran segregation, two genetic crosses were performed to measure the mutants, this does not affect biorientation or segregation of frequency of X-chromosome nondisjunction. These experiments chiasmate chromosomes. measured the frequency of chiasmate and achiasmate chromosome The achiasmate system in Drosophila females efficiently nondisjunction. Segregation of chiasmate chromosomes was segregates homologous chromosomes lacking a crossover measured in females homozygous for normal sequence (Hawley and Theurkauf, 1993). For example, in a female 3802 Journal of Cell Science 124 (22)

Fig. 3. Colocalization of RanT24N with RCC1, and RanQ69L with RanGAP. Wild-type and mutant variants of Ran were detected using an antibody to the HA tag. In all images, Ran, tagged with HA, is in red, RCC1 or RanGAP are in green and DNA is in blue. (A)Inran+ oocytes, Ran and RanGAP appear to be closely associated, but examination of individual optical sections shows they do not colocalize. (B)InranT24N oocytes, RanT24N and RCC1 colocalize around the karyosome. (C)InranQ69L oocytes, RanQ69L and RanGAP colocalize in many clusters throughout the cytoplasm. The arrow points to the karyosome. Scale bars: 10 mm. Journal of Cell Science

heterozygous for a balancer, crossing over is drastically reduced disorganized (Table 3). Despite the similar spindle phenotype, between the homologs. The effect of ranT24N on the achiasmate RanQ69L protein had a localization pattern in oocytes that was system was tested in heterozygotes for the X-chromosomes strikingly different from Ran+ or RanT24N. Whereas Ran and balancer Bwinscy. In contrast to the ranT24N females with normal RanGAP do not colocalize in wild-type oocytes, RanQ69L protein sequence X-chromosomes, Bwinscy heterozygous females showed and RanGAP colocalized in clusters throughout the ranQ69L oocyte a high frequency of X-chromosome nondisjunction (Table 2). (Fig. 3C), suggesting that RanQ69L could be locked in an These results were confirmed with FISH experiments using interaction with RanGAP. Expression of ranQ69L also changed heterochromatic probes to detect centromere orientation. In the the localization pattern of other proteins, such as nuclear Lamin presence of Bwinscy, expression of ranT24N caused orientation (supplementary material Fig. S2). These observations suggest that defects of the X-chromosome but not an autosome (Fig. 5). the expression of RanQ69L in the oocyte affects how nuclear Spindle morphology was similar to that of the ranT24N oocytes envelope proteins interact with the Ran pathway. with normal X chromosomes. These results indicate that in ranT24N mutant oocytes, the achiasmate system of chromosome Maternal expression of ranT24N blocks pronuclear fusion segregation is disrupted. and embryogenesis The spindle organization and chromosome segregation defects Ran locked in the GTP form does not promote we observed in dominant ran mutants were unlikely to be the spindle assembly cause of the sterility in ranT24N females. To determine whether High levels of RanGTP will induce chromatin-independent spindle the low fertility of the ranT24N mutant females was due to assembly in Xenopus oocytes (Carazo-Salas et al., 1999). By an embryonic defect, we examined embryos from mothers contrast, the expression of ranQ69L did not result in the formation expressing the mutant versions of ran. Expression of ran+ of ectopic spindles in the oocyte, as would be expected if RanGTP resulted in zygotes that underwent normal synchronous divisions, is sufficient to initiate spindle assembly (Fig. 2D). Instead, with spindle assembly and chromosome organization expression of ranQ69L in oocytes caused abnormal spindle characteristic of wild-type embryonic divisions (Fig. 6A). assembly reminiscent of ranT24N oocytes; they failed to properly Furthermore, Ran+ localized to the mitotic spindle. This pattern taper microtubules at the poles (Fig. 2C) and the karyosome was of localization is similar to that found in neuroblasts, and Meiotic spindle assembly 3803

Fig. 4. Localization of Mars and TACC to the meiotic spindle is defective in ranT24N mutant oocytes. Mars or TACC-GFP staining (P{[w+]5Ubi-tacc.GFP}1) (Gergely et al., 2000) is in red, DNA is in blue and tubulin is in green except in G where HA is in green. (A)Inran+ oocytes Mars localizes to most microtubules in the meiotic spindle, with the possible exception of the poles and the central spindle. (B–D) The localization of Mars in ranT24N mutant oocytes falls into three categories: present (B), completely absent (C) and reduced (D). (E,F) In wild-type, TACC localizes at the poles and is more concentrated towards the poles. The arrows point to the gap between the TACC staining and the chromosomes. (G,H)InranT24N oocytes, TACC is not restricted to the poles. The arrows point to where the TACC staining meets the chromosomes. Scale bars: 10 mm. Journal of Cell Science

Fig. 5. FISH on Bwinscy/+ oocytes. (A–D) Oocytes in a wild-type background (A); expressing wild-type ran (B); and expressing ranT24N(C,D). Hybridization was performed using probes that bind to highly repeated sequences in the centromeric heterochromatin region of both the X (red) and third (not shown) chromosomes. Tubulin is in green and DNA is in blue. Proper orientation of the homologous chromosomes was scored as the separation of two FISH signals, one signal on each half of the spindle, towards opposite poles. The data are summarized in the Table below. Scale bars: 10. 3804 Journal of Cell Science 124 (22)

Fig. 6. Ran is required to initiate embryonic development. DNA is in blue, Ran tagged with the HA epitope is in red, tubulin is in green, (A) Mitotic spindles form normally when wild-type Ran is expressed. (B) Embryogenesis is blocked when RanT24N is expressed. The presence of a separate male pronucleus (arrow) and no organized microtubules indicates pronuclear fusion has not occurred. The cluster of four DNA masses is the female pronuclei, three of which normally fuse into a polar body. (C) Embryogenesis is blocked when RanQ69L is expressed. These embryos contain a disorganized mass of DNA and tubulin. All transgenes were under the control of the UASP promoter and were expressed in zygotes using the P{GAL4::VP-nos.UTR}MVD1 driver. Scale bars: 10 mm. Journal of Cell Science previously by the injection of fluorescently labeled Ran protein the female and male pronuclei. The failure to observe any nuclei in into embryos (Trieselmann and Wilde, 2002). Thus, expression half the embryos could indicate a failure to reform the nuclear of ran+ produces no deleterious effects on the mitotic divisions of envelope following completion of meiosis (Ciciarello et al., 2007) the embryo and recapitulates the known localization pattern to or a failure of pronuclear fusion. spindle microtubules. Similar to the effect of ranT24N, maternal expression of ranQ69L The majority of zygotes expressing ranT24N arrested led to a failure to initiate embryonic development. In most of the development without any evidence of the embryonic mitotic mutant zygotes, a cluster of DNA and microtubules was observed divisions. In wild-type zygotes, the two meiotic divisions are in the center of the cell and there was no evidence of any mitotic completed without the formation of polar bodies (Demerec, 1950). divisions (Fig. 6C). This phenotype was different from the two Therefore, prior to pronuclear fusion, the oocyte contains four observed with ranT24N and might be due to a defect shortly after female meiotic products and the sperm nucleus. Three of the pronuclear fusion (see Discussion). female products fuse, while the fourth fuses with the sperm nucleus. The ranT24N zygotes were of two types. Approximately ran is required for cytoplasmic microtubule assembly half of them contained the unfused female and male meiotic The lack of pronuclear fusion in the ranT24N mutant suggests products and were devoid of organized microtubules (Fig. 6B). In that RanGTP is required for microtubule assembly that occurs in these cases, RanT24N protein was closely associated with the the cytoplasm. To test this possibility, we determined whether chromosomes, as would be expected if it was bound to RCC1. The RanGTP has a role in another example of microtubule assembly remaining half of the zygotes had no visible nuclei. These results that does not involve direct interactions with the chromatin. suggest that meiosis can be completed in the ranT24N zygotes but Such ‘cytoplasmic microtubule assembly’ occurs in Drosophila the female and male pronuclei do not fuse and the three remaining oocytes expressing a mutation in subito (sub) that removes the female meiotic products fail to aggregate. Consistent with this N-terminal domain of the protein. As observed previously, conclusion, we have observed normal meiosis II spindles in expression of P{UASP:subDNT} resulted in the formation of ranT24N embryos (data not shown). Expression of ranT24N in ectopic spindles in the oocyte (Table 4; Fig. 7) (Jang et al., embryos appears to disrupt the assembly of the microtubule 2007). These ectopic spindles do not form until after NEB, network nucleated by the sperm centrosome that brings together consistent with a diffusible nuclear factor being required for Meiotic spindle assembly 3805

Table 4. Characterization of ectopic spindle phenotype in subDNT double mutants Genotype Total oocytes Oocytes containing ectopic spindles Ectopic spindle (%)a subDNT 93 91 98 subDNT; ran+ 63 61 97 ranT24N 16 0 0 subDNT; ranT24N 28 0 0

Each genotype was expressed by crossing to P{GAL4::VP16-nos.UTR}MVD1 driver. aEctopic spindle (%) is equal to the number of oocytes with ectopic spindles divided by the total number of oocytes.

their formation. Ectopic spindles cluster and form in many because it is present in many clusters, possibly vesicles, within regions of the mutant oocytes without direct contact with the oocyte, suggesting that conversion of RanGTP to RanGDP chromosomes. might be regulated and only occur in certain locations. This could We tested whether RanGTP in the cytoplasm stimulates mean that a gradient of RanGTP (Clarke and Zhang, 2008; Kalab microtubule assembly by constructing a double mutant with the and Heald, 2008) is not established in the oocyte. A candidate N-terminal deletion mutation using subDNT and ranT24N. protein responsible for generating the concentrations of RanGAP Typically, two to four clusters of ectopic spindles could be is Ran binding protein 2 (RanBP2; also known as Nup358). This observed in subDNT mutant oocytes, such as at the posterior tip protein is found within the nuclear envelope and binds to and the region near the karyosome (Fig. 7A,B). Any oocyte RanGAP (Hutten et al., 2008). Following NEB, RanGAP could containing more than one cluster of spindle formation was be anchored to RanBP2-containing cytoplasmic vesicles. considered to have the ectopic spindle phenotype. The frequency Ran has an unusual localization pattern in oocytes; of subDNT oocytes expressing ran+ with ectopic spindles was concentrating around the outside of the spindle. By contrast, similar to that with subDNT alone (96.8% and 97.9%, respectively; Ran overlaps with the spindle in Drosophila mitotic cells (this Table 4). Strikingly, the dominant-negative mutation ranT24N work) (Silverman-Gavrila and Wilde, 2006; Trieselmann and completely suppressed the ectopic spindle phenotype (Fig. 7C,D; Wilde, 2002). We have not determined whether these Table 4). The only spindle that formed in ranT24N; subDNT concentrations of Ran are in the GDP or GTP state. However, oocytes was around the karyosome. These results suggest that we can speculate on the basis of the localization patterns of wild- RanGTP is required for the interaction between SubitoDNT and type and mutant proteins. From this type of evidence, microtubules that occurs in the absence of the chromosomes. In Trieselmann and Wilde suggested that the bulk of Ran on the other words, RanGTP could be required for microtubule embryonic spindle is in the GTP state. (Trieselmann and Wilde, assembly that does not depend on direct contacts with the 2002) Similarly, the bulk of the Ran localized around the outside chromosomes. Another surprising finding was that the of the meiotic spindle might be in the GTP form. The pattern of suppression was reciprocal. The ranT24N; subDNT double mutant mutant RanQ69L staining suggests it enters RanGAP-containing had increased fertility relative to the two single mutants (Table 2) vesicles but does not leave because it is not hydrolyzed. Thus, the Journal of Cell Science and this correlated with an increased frequency of embryos wild-type Ran that localizes adjacent to the clusters of RanGAP undergoing mitosis (Fig. 7E). could be the GDP form of the protein that has left RanGAP- containing vesicles. Discussion The Ran pathway has a variety of targets, which leads to effects RanGTP has a role in organizing the acentrosomal on kinetochores, centrosomes and microtubule-associated spindle poles proteins (Kalab and Heald, 2008). RanGTP is potentially an RCC1 and RanGTP were found to be required for chromatin- important molecule for spindle assembly in acentrosomal oocytes induced spindle assembly in Xenopus extracts (Carazo-Salas because it has been identified as a key factor for chromatin- et al., 1999; Kalab et al., 1999). In such extracts, expression of induced spindle formation in Xenopus extracts (Carazo-Salas RanT24N blocks spindle assembly (Ohba et al., 1999) and high et al., 1999; Kalab et al., 1999; Karsenti and Vernos, 2001; Ohba concentrations of RCC1 or expression of a GTP-locked form of et al., 1999). Surprisingly, our results suggest that RanGTP might Ran leads to spindle formation in the absence of chromosomes be more important for microtubule assembly in other and centrosomes (Carazo-Salas et al., 1999). circumstances, such as when centrosomes are present or when Our analysis of RanGTP function in Drosophila oocytes is microtubules assemble without direct contact with the based on these and numerous other studies in which expression of chromosomes. the ranT24N mutation effectively reduces the concentration of RanGTP. We believe that the ranT24N mutant had the desired Regulators of RanGTP, RCC1 and RanGAP, during meiosis effect of reducing RanGTP production, for four reasons. First, in Drosophila females expression of RanT24N in somatic cells caused embryonic or early Diffusion of RanGTP from its source, the chromatin, into the larval lethality. Second, RanT24N localized tightly to the meiotic cytoplasm, where it is converted into RanGDP, can create a chromosomes, consistent with the expectation that this form of gradient that regulates microtubule organization (Caudron et al., Ran remains bound to RCC1 because it has a low rate of GTP 2005; Kalab et al., 2006). Drosophila oocytes contain two key exchange. The high affinity of RanT24N for RCC1 causes a regulators of the Ran pathway in distinct locations. RCC1, as reduction in the production of RanGTP (Dasso et al., 1994). expected, is located tightly around the karyosome in mature Third, the spindle organization defects observed in ranT24N oocytes. RanGAP localization is more complex than expected oocytes were similar to defects seen in mars1 mutant oocytes, a 3806 Journal of Cell Science 124 (22) Journal of Cell Science

Fig. 7. Ran is required for ectopic spindle formation caused by subDNT, a mutation that deletes the N-terminal non-motor domain of the kinesin 6, Subito. In all the images, SubitoDNT is a GFP fusion protein and is shown in red, tubulin is in green and DNA is in blue. (A) Ectopic spindles form in subDNT oocytes expressing Ran+. This is an example where several spindles have formed nears the chromosomes. (B) Low magnification image of ectopic spindles in subDNT oocytes expressing Ran+. There are two clusters of ectopic spindles in the oocyte. (C) Expression of RanT24N in oocytes suppresses the ectopic spindle phenotype of subDNT. There are several masses of DNA in the subDNT single mutant but only a single karyosome and spindle in the double mutant. (D) A low magnification image of the same oocyte shown in B. The arrow in D points to the chromosomes and spindle. (E) subDNT suppresses the arrest in embryonic development caused by ranT24N. Scale bars: 10 mm.

protein known to be regulated by the Ran pathway. Fourth, conclude that the reduction in RanGTP levels sufficient to block ranT24N caused dramatic disruptions in chromosome-independent pronuclear fusion were not sufficient to block acentrosomal microtubule assembly assays, such as pronuclear fusion (see spindle assembly. below). Because these chromosome-dependent (meiotic spindle) Unlike the results in Xenopus extracts, expression of the and -independent functions occur in the same cytoplasm, we dominant-negative GDP-locked variant of Ran had relatively Meiotic spindle assembly 3807

mild effects on Drosophila oocyte spindle assembly and assembly is not known, the ranQ69L mutation might cause defects in karyosome organization. Meiosis I spindles were bipolar in membranous structures that have a role in spindle organization. We ranT24N oocytes. Indeed, reducing the RanGTP concentration in have found that Ran and Axs are closely associated, although at the the oocyte was not sufficient to severely affect either meiotic light microscope level it is difficult to determine if Ran is inside or division, because meiosis II spindles (data not shown) and female outside the Axs staining (J.C. and K.S.M., unpublished results). meiotic products could be seen in the embryos. The most Similar to the oocytes, the phenotype of the ranQ69L mutant important defect was that the meiosis I spindle often had non- zygotes might be associated with defects in membrane structure. tapered poles, and was associated with the abnormal localization In ranQ69L mutants, a single cluster of DNA and microtubules of proteins necessary for pole formation such as Mars or TACC could be observed in the center of the zygote. A strikingly similar (Cullen and Ohkura, 2001). These results are consistent with phenotype has been observed in dominant-negative Ketel experiments in embryos that found depletion of RanGTP causes mutants; Ketel is the Drosophila homolog of importin-b defects in spindle pole organization and chromosome (Timinszky et al., 2002; Tirian et al., 2000). In the Ketel organization and congression (Silverman-Gavrila and Wilde, dominant mutants, meiosis I and II occur and the female and 2006). Abnormal spindle morphology in oocytes could be the male pronuclei come together, but they interact abnormally reason for the disorganized karyosome phenotype and because of defects in the nuclear envelopes. Subsequently, the nondisjunction of achiasmate chromosomes. Loss of RanGTP first mitotic division fails and the chromosomes disintegrate could result in a failure to activate Aurora A, which within a large aggregate of microtubules. Similar to the Ketel phosphorylates TACC (Barros et al., 2005; Kalab and Heald, mutant, ranQ69L could cause abnormal interactions among 2008). In embryos, TACC localization to the centrosomes nuclear envelope proteins in the zygote, causing a failure in the depends on phosphorylation by Aurora A (Barros et al., 2005). first mitotic division. TACC initially binds all microtubules, but as the spindle matures, TACC is phosphorylated and localizes to the poles. Expression of RanGTP is required for achiasmate ranT24N in oocytes might cause a reduction in Aurora A activity, chromosome segregation resulting in a failure to phosphorylate TACC and localize it to the There are two chromosome segregation mechanisms in poles. Further studies are needed, however, because the role of Drosophila females. The first is the segregation of bivalents Aurora A in Drosophila female meiosis is not known. In addition, connected by chiasmata (Hawley, 1988), which is how most Mars might have a role in promoting the dephosphorylation of chromosomes segregate. The second is of the chromosomes that TACC (Tan et al., 2008). Overall, RanGTP might have a specific lack chiasmata. This includes the small fourth chromosome, role in organizing spindle poles but might not be required for which always lacks crossovers, and larger chromosomes, which chromosome-promoted spindle assembly in oocytes. lack a crossover in approximately 5% of meioses. Homologous Expression of ranT24N did not block spindle assembly. pairs can be forced into the achiasmate system with balancers that Conversely, expression of the GTP-locked mutant, ranQ69L, did suppress crossing over. In all these cases, homologous not induce an uncoupling between spindle assembly and the chromosomes segregate correctly even though they are not chromosomes, as it does in Xenopus oocytes (Carazo-Salas et al., connected by chiasmata. Expression of ranT24N had only mild

Journal of Cell Science 1999). Thus, RanGTP might not be sufficient to initiate spindle effects on chiasmate segregation, but had a severe effect on the assembly in Drosophila oocytes. Surprisingly, ranQ69L oocytes segregation of achiasmate X-chromosomes. These results suggest showed loss-of-function spindle phenotypes similar to ranT24N that the spindle pole organization defects caused by low RanGTP mutant oocytes. Interestingly, manipulation of RanGTP levels levels affect chromosome segregation. with T24N or Q69L mutations in mammals has similar phenotypes. For example, the expression of either form of Ran RanGTP is required for chromosome independent in mouse oocytes resulted in similar meiosis II spindle microtubule assembly phenotypes (Dumont et al., 2007). These results suggest that Unlike assembly of the meiosis I spindle, expression of ranT24N the effects of manipulating RanGTP levels in an intact oocyte are blocked two other types of microtubule assembly. First, ranT24N not easily predicted by experiments in Xenopus extracts. Other mutants had a defect in the fusion of the female and male factors such as protein localization might play important roles in pronuclei. This was the most probable cause of the fertility regulating the Ran pathway. We also cannot rule out the defect in ranT24N mutants. Several genes with roles in possibility that the Ran pathway functions differently in oocyte microtubule assembly are also required for pronuclear fusion, meiosis, such as if active Ran is not GTP dependent. including subito (Giunta et al., 2002). This process depends on We suggest there could be two reasons for the similarity of the the assembly of a microtubule array that is nucleated by the ranQ69L and ranT24N phenotypes. First, expressing the GTP- centrosome donated by the sperm, and acts to draw the female locked ranQ69L mutation can inhibit the binding of RCC1 to the pronucleus towards the male pronucleus. Second, ranT24N chromatin (Zhang et al., 2002), causing a reduction in RanGTP suppressed the formation of the ectopic spindles that form in a near the chromatin. Alternatively, the phenotypes of the ranQ69L neomorphic subito mutant (subDNT) (Jang et al., 2007). The mutant oocyte might be associated with defects in the formation of these spindles occurs after NEB, consistent with a organization of membranes or vesicles. For example, dependence on release of RanGTP from the nucleus. Both of expression of the ranQ69L mutation caused Lamin, RanGAP and these examples involve assembly of microtubules without direct RanQ69L to colocalize in globular structures throughout the interaction with the chromosomes and suggest that the assembly oocyte. Kramer and Hawley (Kramer and Hawley, 2003) have and bundling of microtubules in the oocyte cytoplasm depend on proposed that the transmembrane protein Axs is a component of a RanGTP. membranous structure surrounding the meiotic spindle. With the One characteristic of the ectopic spindles in subDNT mutants is caveat that the link between membranous structures and spindle that they form in discrete clusters within the oocyte. Because 3808 Journal of Cell Science 124 (22)

RanGAP appears in clusters, it is possible that RanGTP is not in a into the pPHW vector that encodes three copies of the HA epitope at the N-terminus of the coding region in a pUASP backbone (Rorth, 1998). Amino acid gradient or distributed evenly in the cytoplasm. Thus, an substitutions were made by modifying the wild-type ran clone in pENTR4 using interesting possibility is that the regions containing ectopic the Change-IT mutagenesis kit (USB) and the appropriate primers. For the ranT24N spindles are where the concentration of RanGTP-dependent transgene, an asparagine was substituted for a threonine at amino acid 24. For the Q69L spindle assembly factors are at their highest. ran transgene, a leucine was substituted for a glutamine at amino acid 69. For ubiquitous expression in somatic tissues, males carrying a ran transgene, These experiments revealed a surprising mutual suppression by P{UASP::ran}, were crossed to females carrying a GAL4 transgene with a D the sub NT and ranT24N mutations. Although both mutants have tubulin promoter (P{tubP-GAL4}) (Lee and Luo, 1999). A cross with the driver decreased fertility, the double mutant is fertile. One interpretation heterozygous to a balancer that provides a Tubby phenotype visible in larvae, DNT P{tubP-GAL4}/T(2;3)B3, CyO: TM6B, Tb, results in two genotypes: is that RanGTP regulates Subito, and the sub mutation P{UASP::ran}/P{tubP-GAL4} and P{UASP::ran}/T(2;3)B3, CyO: TM6B, Tb. bypasses the dependence on RanGTP. However, we found that The percentage survival was calculated as (Tb+ flies)/(total flies). For expression ran mutations did not affect Subito localization or the formation in the germline and early embryo, males carrying a ran transgene were crossed to of the central spindle. A more probable explanation is that there females carrying a GAL4 transgene with a nanos promoter, P{GAL4::VP16- nos.UTR}MVD1 (Van Doren et al., 1998). To measure fertility and chromosome are two independent spindle assembly pathways in the oocyte and segregation during meiosis, females carrying a transgene and the nanos driver the loss of spindle assembly factors in ranT24N zygotes is were crossed to either yw/BSY or C(1;Y)v f B, c(4) ci eyR males. The S + + S S balanced by the enhanced spindle assembly activity present in the nondisjunction frequency was calculated as 2(B R+B =)/[B R+B =+2(B R+B+ =)]. subDNT mutant. Expression of ranT24N might suppress the sterility DNT phenotype of sub by abolishing ectopic spindles, whereas Antibodies and immunofluorescence microscopy DNT T24N sub might suppress the reduced fertility phenotype of ran Mature (stage 14) oocytes were collected from 50–200 yeast-fed females that were by overcoming the defects in microtubule assembly needed for aged 3–4 days by physical disruption in a common household blender (McKim processes such as pro-nuclear fusion. et al., 2009; Theurkauf and Hawley, 1992). The oocytes were fixed in modified Robb’s medium and cacodylate–formaldehyde fixative for 8 minutes and then their outer membranes were removed by rolling the oocytes between the frosted part of Conclusions a slide and a coverslip. Using dominant-negative mutations,wehavefoundthatRanGTPis Embryos were collected by placing females and males in cages with grape juice plates for 2 hours to enrich for those undergoing the syncytial divisions. Embryos required for the fertility of Drosophila females. We found no evidence were removed from the grape juice plates with water and placed in 50% bleach for that RanGTP is required, or sufficient, for the initiation of acentrosomal 90 seconds to remove the chorion. They were then thoroughly washed with water spindle assembly in Drosophila oocytes. We did detect a role in to remove all traces of bleach. The embryos were fixed using heptane and organizing the spindle poles that could be explained by RanGTP methanol (Rothwell and Sullivan, 2000). For squashed neuroblast preparations, the third instar larval brains were regulation of proteins such as Mars/Hurp, TACC and Aurora A. These dissected in saline and the brains were fixed in 3.7% formaldehyde in 16 PBS for defects, however, would not be expected to have a severe effect on 30 minutes. The brains were then placed in 45% acetic acid for 3 minutes before fertility. A similar conclusion was drawn from expressing a dominant- transferring to ,8 ml of 60% acetic acid on a siliconized coverslip where they were firmly squashed between the coverslip and slide. The slides were briefly negative form of Ran in mouse oocytes or when RCC1 was depleted frozen in liquid nitrogen and the coverslips were flicked off with a razor blade. The from Xenopus oocytes (Dumont et al., 2007). By analyzing mutants slides were placed in ethanol at 220˚C (chilled on dry ice) for 10 minutes, then similar to the ones we used here, only mild defects in meiosis I spindle transferred to a slide chamber containing 0.1% Triton X-100 in PBS for 10 minutes. Rubber cement was used to form wells on the slides and the preparations assembly were found, such as a delay establishing bipolarity. The were washed twice for 5 minutes each with PBS. The tissue was blocked with 1%

Journal of Cell Science failure to observe evidence supporting a role for RanGTP in BSA in PBS for 45 minutes. acentrosomal spindle assembly might be explained by a predominant Oocytes, embryos and neuroblasts were stained for DNA with Hoechst 33342 at chromosome-dependent pathway in oocytes involving the a 1:1000 dilution (10 mg/ml solution) and for microtubules with mouse anti-a- tubulin monoclonal antibody DM1A (1:50), directly conjugated to FITC (Sigma) chromosome passenger complex (CPC). The CPC is required for or rat anti-a-tubulin monoclonal antibody (1:75; Millipore). The primary chromosome-dependent spindle assembly in Xenopus egg extracts antibodies were rat anti-SUB antibody (used at 1:75) (Jang et al., 2005), rat (Maresca et al., 2009; Sampath et al., 2004) and Drosophila oocytes anti-HA (Roche, clone 3F10: 1:25), rat anti-INCENP (1:500) (Wu et al., 2008), (Colombie et al., 2008) (S. Radford and K.S.M., unpublished results). mouse anti-RCC1 (1:20) (Frasch, 1991), rabbit anti-RanGAP (1:800) (Kusano et al., 2001), rabbit anti-Mars (Tan et al., 2008) and mouse anti-Lamin Dm0 Our results suggest that, compared with chromosome-mediated (1:800) (Klapper et al., 1997). These primary antibodies were detected with either spindle assembly, RanGTP has a greater role in microtubule a Cy3 or Cy5 secondary antibody preabsorbed against a range of mammalian organization when centrosomes are present or when at a distance serum proteins (Jackson Labs) and Drosophila embryos. TACC was detected using a GFP fusion protein (Gergely et al., 2000). Images were collected on a Leica TCS from the chromosomes. We suggest that NEB preceding the SP2 confocal microscope with a 636, 1.3 NA lens. Images are maximum assembly of the meiosis I spindle releases RanGTP into the projections of image stacks with the individual channels merged and then cropped cytoplasm, which results in a cytoplasm enriched for active in Adobe Photoshop. spindle assembly factors. The restriction of RanGAP to vesicle- Fluorescent in situ hybridization like structures could leave a considerable amount of RanGTP in Stage 14 oocytes were collected as described above and then processed for both the cytoplasm. This activity has only a minor role in meiotic immunofluorescence and fluorescent in situ hybridization (FISH) as described spindle assembly, but is crucial for the early events of previously (Dernburg, 2000; McKim et al., 2009). Oligonucleotides embryogenesis. The microtubule array facilitating pronuclear corresponding to the satellite sequence AACAC for the second chromosome centric heterochromatin or CCCGTACTCGGT (Dodeca) for the third fusion assembles while the nuclear envelope is intact. Therefore, chromosome centric heterochromatin were end-labeled with Cc3-dCTP or Cc5- the oocyte might accumulate and store RanGTP during the dCTP (GE Healthcare) by Terminal Deoxynucleotidyl Transferase (Invitrogen). meiotic divisions when there is no nuclear envelope in order to A probe to the 359 bp repeat on the X-chromosome was amplified by PCR and end labeled as described previously (Dernburg, 2000). Oocytes were support pronuclear fusion, when the nuclear envelope is intact. subsequently stained for microtubules and DNA as described above.

Materials and Methods Acknowledgements Generation and analysis of transgenic lines We are grateful to Li Nguyen for technical assistance, Daimark Full-length and substitution derivatives of ran were amplified by PCR. The clones 1 were verified by sequencing and then the fragments were cloned into the pENTR4 Bennett for providing Mars antibodies and the mars mutant and vector (Gateway). The fragment was then recombined using Clonase (Invitrogen) Janet Jang for Fig. 2A. Some stocks used in this study were obtained Meiotic spindle assembly 3809

from the Bloomington Stock Center and some antibodies were from Hawley, R. S. and Theurkauf, W. E. (1993). Requiem for distributive segregation: Developmental Studies Hybridoma Bank at the University of Iowa, Achiasmate segregation in Drosophila females. Trends Genet. 9, 310-317. Hutten, S., Flotho, A., Melchior, F. and Kehlenbach, R. H. (2008). The Nup358- developed under the auspices of the National Institute of Child RanGAP complex is required for efficient importin alpha/beta-dependent nuclear Health and Human Development. import. Mol. Biol. Cell 19, 2300-2310. Jang, J. K., Rahman, T. and McKim, K. S. (2005). The kinesin-like protein Subito Funding contributes to central spindle assembly and organization of the meiotic spindle in Drosophila oocytes. Mol. Biol. Cell 16, 4684-4694. This work was supported by a fellowship from the Busch foundation Jang, J. K., Rahman, T., Kober, V. S., Cesario, J. and McKim, K. S. (2007). 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