Ran Localizes around the Microtubule Spindle In Vivo during Mitosis in Drosophila Embryos (original) (raw)

1 King's College Circle confocal microscopy (LSCM). Between nuclear cycles 9 and 14, the Drosophila embryo is a syncitium in which Toronto, Ontario M5S 1A8 Canada the nuclei localize at the embryo surface, allowing visualization, and injected material can diffuse throughout the embryo, reaching many nuclei and spindles. Summary Mitotic Localization of RCC1 and RanGAP The GTPase Ran regulates multiple cellular functions Central to the model described above is the idea that throughout the cell cycle, including nucleocytoplasmic RCC1 generates RanGTP while associated with chromatransport, nuclear membrane assembly [1], and spintin throughout mitosis. However, the mitotic localization dle assembly [2-10]. Ran mediates spindle assembly of RCC1 is controversial: immunostaining studies in huby affecting multiple spindle assembly pathways: miman tissue culture cells (HeLa S3) and Drosophila emcrotubule dynamics [6, 7], microtubule motor activity bryos found RCC1 distributed throughout the cell in [6], and spindle pole assembly [8-10]. Ran is predicted mitosis [14, 15], whereas, in mitotic Xenopus egg exto facilitate spindle assembly by remaining in the GTPtract, RCC1 bound to DNA-coated beads [5]. Two apbound state around the chromatin in mitosis. Here, proaches were taken here to determine the mitotic localwe directly test the central tenet of this hypothesis in ization of RCC1. First, RCC1 was immunolocalized to vivo by determining the cellular localization of Ran mitotic chromatin in methanol-fixed Drosophila empathway components in Drosophila embryos. We find bryos (Figure 1A). As the major difference between these that, during mitosis, RCC1, the nucleotide exchange findings and those of prior studies is the fixation condifactor for Ran, is associated with chromatin, while Ran tions [14, 15], RCC1 localization was followed by timeand RanL43E, an allele locked in the GTP-bound state, lapse LSCM in vivo in the absence of fixation by injecting localize around the spindle. In contrast, nuclear prorhodamine-labeled RCC1 into GFP-histone-expressing teins redistribute throughout the embryo upon nuclear Drosophila embryos. RCC1 colocalized with GFP-hisenvelope breakdown (NEB). Thus, in vivo RanGTP has tone throughout the cell cycle, indicating that RCC1 was the correct spatial localization within the cell to moduassociated with chromatin during mitosis (Figure 1B). late spindle assembly. Since RCC1 is active in mitotic Xenopus egg extract [3, 5, 8], which implies that, in mitosis, RCC1 is active and capable of generating RanGTP, we infer from the mitotic Results and Discussion localization of RCC1 in Drosophila embryos that RanGTP is generated throughout mitosis at or near the chro-A working model has been proposed to explain the mimatin. totic roles of Ran [8-10]. In interphase, some spindle RanGAP can stimulate the hydrolysis of GTP to form assembly factors (SAFs) are sequestered into the nu-RanGDP. Immunostaining of fixed embryos revealed a cleus through the action of nuclear transport receptors punctate nuclear envelope staining pattern for RanGAP (NTRs), and they only participate in spindle assembly (Figure 1C). Occasionally, the punctate staining pattern upon nuclear envelope breakdown (NEB). However, followed a microtubule on the outer face of the spindle after NEB, these nuclear SAFs (nSAFs) could be inhib-(Figure 1C, arrow). Previously, RanGAP was localized to ited by rebinding to NTRs, an interaction that would be spindles and kinetochores in mammalian tissue culture prevented by RanGTP binding to NTRs. Therefore, it was cells [16, 17]. We did not detect any staining at the postulated that, during mitosis, RanGTP is continually kinetochore. However, this could be due to either a spegenerated at the chromatin to ensure that spindle ascies difference or the epitope recognized by the antisembly occurs around the chromatin. Since RCC1, the body being hidden at the kinetochore [17]. nucleotide exchange factor for Ran [11], can bind to Our finding that RCC1 is bound to chromatin throughchromatin [12], a gradient of RanGTP could exist with out mitosis suggests that RanGTP can be generated at its highest concentration at the chromosomes. or close to the chromatin in mitosis in vivo. In addition, The mitotic localization of RCC1 and RanGTP is cenas RanGAP remains in the residual nuclear envelope, tral to this model. Recently, an elegant study using novel the highest concentration of RanGTP would remain biosensors with different properties in the presence of around the chromatin in mitosis, a finding that supports RanGTP or RanGDP suggested that, in vitro, RanGTP a recent in vitro study [13]. is localized around the chromatin and the spindle [13]. In an alternative approach, we examined the mitotic localization of Ran and Ran pathway components in Ran Localizes around the Spindle in Mitosis vivo. Drosophila embryos were injected between nu-The model predicts that RanGTP remains close to its site of generation at the chromatin throughout mitosis. To analyze the mitotic localization of Ran in vivo, Dro