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Rangtp Is Required for Meiotic Spindle Organization and the Initiation of Embryonic Development in Drosophila

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Rangtp Is Required for Meiotic Spindle Organization and the Initiation of Embryonic Development in Drosophila 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
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