The small guanosine triphosphatase Ran is loaded with guanosine triphosphate (GTP) by the chromatin-bound guanine nucleotide exchange factor RCC1 and releases import cargoes in the nucleus during interphase. In mitosis, Ran-GTP promotes spindle assembly around chromosomes by locally discharging cargoes that regulate microtubule dynamics and organization. We used fluorescence resonance energy transfer-based biosensors to visualize gradients of Ran-GTP and liberated cargoes around chromosomes in mitotic Xenopus egg extracts. Both gradients were required to assemble and maintain spindle structure. During interphase, Ran-GTP was highly enriched in the nucleoplasm, and a steep concentration difference between nuclear and cytoplasmic Ran-GTP was established, providing evidence for a Ran-GTP gradient surrounding chromosomes throughout the cell cycle.

The RanGTPase cycle provides directionality to nucleocytoplasmic transport, regulating interactions between cargoes and nuclear transport receptors of the importin-beta family. The Ran-importin-beta system also functions in mitotic spindle assembly and nuclear pore and nuclear envelope formation. The common principle underlying these diverse functions throughout the cell cycle is thought to be anisotropy of the distribution of RanGTP (the RanGTP gradient), driven by the chromatin-associated guanine nucleotide exchange factor RCC1 (refs 1, 4, 5). However, the existence and function of a RanGTP gradient during mitosis in cells is unclear. Here we examine the Ran-importin-beta system in cells by conventional and fluorescence lifetime microscopy using a biosensor, termed Rango, that increases its fluorescence resonance energy transfer signal when released from importin-beta by RanGTP. Rango is predominantly free in mitotic cells, but is further liberated around mitotic chromatin. In vitro experiments and modelling show that this localized increase of free cargoes corresponds to changes in RanGTP concentration sufficient to stabilize microtubules in extracts. In cells, the Ran-importin-beta-cargo gradient kinetically promotes spindle formation but is largely dispensable once the spindle has been established. Consistent with previous reports, we observe that the Ran system also affects spindle pole formation and chromosome congression in vivo. Our results demonstrate that conserved Ran-regulated pathways are involved in multiple, parallel processes required for spindle function, but that their relative contribution differs in chromatin- versus centrosome/kinetochore-driven spindle assembly systems.

mitotic spindle during mitosis. Through exportin 1, RanGTP recruits essential centrosome and kinetochore components, whereas the RanGTP-induced release of spindle assembly factors (SAFs) from importins activates SAFs to nucleate, bind and organize nascent spindle microtubules. Although a considerable fraction of cytoplasmic SAFs is active and RanGTP induces only partial further activation near chromatin, bipolar spindle assembly is robustly induced by cooperativity and positive-feedback mechanisms within the network of Ranactivated SAFs. The RanGTP gradient is conserved, although its roles vary among different cell types and species, and much remains to be learned regarding its functions. Journal of Cell Science 1578 (Hutchins et al., 2004;Li and Zheng, 2004a). Both modes of RCC1-chromatin interaction are required for correct spindle assembly in tissue culture cells (Chen et al., 2007; Hutchins et al., 2004;Li and Zheng, 2004a) and probably function simultaneously to strengthen the interaction of RCC1 with chromatin during the nucleotideexchange reaction (Chen et al., 2007). Ran binds directly to histones H3 and H4 on chromatin (Bilbao-Cortes et al., 2002), promoting its interaction with RCC1. Fluorescence recovery after photobleaching experiments in live cells indicate that cooperative binding of RanGDP and RCC1 to chromatin is followed by the release of RanGTP after the completion of GDP/GTP exchange (Li and Zheng, 2004b), thereby heightening the peak of the RanGTP gradient and focusing it to a thin volume at the interface of chromatin and cytoplasm.Interestingly, the histone-binding domain of RCC1 overlaps with its NLS, which is recognized by a complex between importin α3 and importin β that imports RCC1 into the nucleus during interphase. In mitosis, bound importins can block the interaction between RCC1 and chromatin in the absence of mitotic cyclindependent kinase 1 (Cdk1)-dependent phosphorylation of serine residues that are adjacent to the NLS (Hutchins et al., 2004;Li and Zheng, 2004a). However, human cells express three isoforms of RCC1 with variations in their N-termini that impose strikingly different regulation by phosphorylation and importins. Only RCC1γ is a substrate of mitotic kinases, but it does not bind well to importins; conversely, RCC1α is not mitotically phosphorylated and binds to importins well, indicating that these two isoforms may be specialized for mitotic and interphase functions, respectively (Hood Journal of Cell Science 121 (10) Clarke, 2007). In addition, all the isoforms are expressed in a complex tissue-specific manner (Hood and Clarke, 2007). Clearly, much remains to be learned about RCC1 function and regulation. Journal of Cell Science1579 Mitotic RanGTP gradient and Cytoplasmic RanGAP catalyzes the conversion of RanGTP to RanGDPAfter RCC1-catalyzed nucleotide exchange, RanGTP that diffuses from the chromatin can either be rapidly converted to RanGDP by RanGAP, or bind to abundant NTRs in the cytoplasm. RanGTP that is bound to an NTR is protected from RanGAP until it is ex...

Spindle formation is essential for stable inheritance of genetic material. Experiments in various systems indicate that Ran GTPase is crucial for meiotic and mitotic spindle assembly. Such an important role for Ran in chromatin-induced spindle assembly was initially demonstrated in Xenopus laevis egg extracts. However, the requirement of RanGTP in living meiotic cells has not been shown. In this study, we used a fluorescence resonance energy transfer probe to measure RanGTP-regulated release of importin β. A RanGTP-regulated gradient was established during meiosis I and was centered on chromosomes throughout mouse meiotic maturation. Manipulating levels of RanGTP in mice and X. laevis oocytes did not inhibit assembly of functional meiosis I spindles. However, meiosis II spindle assembly did not tolerate changes in the level of RanGTP in both species. These findings suggest that a mechanism common to vertebrates promotes meiosis I spindle formation in the absence of chromatin-induced microtubule production and centriole-based microtubule organizing centers.

Ran is an abundant nuclear GTPase with a clear role in nuclear transport during interphase but with roles in mitotic regulation that are less well understood. The nucleotide-binding state of Ran is regulated by a GTPase activating protein, RanGAP1, and by a guanine nucleotide exchange factor, RCC1. Ran also interacts with a guanine nucleotide dissociation inhibitor, RanBP1. RanBP1 has a high affinity for GTP-bound Ran, and it acts as a cofactor for RanGAP1, increasing the rate of GAP-mediated GTP hydrolysis on Ran approximately tenfold. RanBP1 levels oscillate during the cell cycle [4], and increased concentrations of RanBP1 prolong mitosis in mammalian cells and in Xenopus egg extracts (our unpublished observations). We investigated how increased concentrations of RanBP1 disturb mitosis. We found that spindle assembly is dramatically disrupted when exogenous RanBP1 is added to M phase Xenopus egg extracts. We present evidence that the role of Ran in spindle assembly is independent of nuclear transport and is probably mediated through changes in microtubule dynamics.

During interphase, the transport receptor importin-β carries cargoes into the nucleus, where RanGTP releases them. A similar mechanism operates in mitosis to generate a gradient of active spindle assembly factors around mitotic chromosomes. Importin-β and RanGTP have been implicated in additional cellular processes, but the precise roles of the Ran/importin-β pathway throughout the cell cycle remain poorly understood. We implemented a FRET-based, high-throughput small molecule screen for compounds that interfere with the interaction between RanGTP and importin-β and identified importazole, a 2,4-diaminoquinazoline. Importazole specifically blocks importin-β-mediated nuclear import both in Xenopus egg extracts and cultured cells, without disrupting transportin-mediated nuclear import or CRM1-mediated nuclear export. When added during mitosis, importazole impairs the release of an importin-β cargo FRET probe and causes both predicted and novel defects in spindle assembly. Together, these results indicate that importazole specifically inhibits the function of importin-β, likely by altering its interaction with RanGTP. Importazole is a valuable tool to evaluate the function of importin-β/RanGTP pathway at specific stages during the cell cycle.

Before fertilization can occur, mammalian sperm must undergo capacitation, a process that requires a cyclic AMP-dependent increase in tyrosine phosphorylation. To identify proteins phosphorylated during capacitation, two-dimensional gel analysis coupled to antiphosphotyrosine immunoblots and tandem mass spectrometry (MS/MS) was performed. Among the protein targets, valosin-containing protein (VCP), a homolog of the SNARE-interacting protein NSF, and two members of the A kinase-anchoring protein (AKAP) family were found to be tyrosine phosphorylated during capacitation. In addition, immobilized metal affinity chromatography was used to investigate phosphorylation sites in whole protein digests from capacitated human sperm. To increase this chromatographic selectivity for phosphopeptides, acidic residues in peptide digests were converted to their respective methyl esters before affinity chromatography. More than 60 phosphorylated sequences were then mapped by MS/MS, including precise sites of tyrosine and serine phosphorylation of the sperm tail proteins AKAP-3 and AKAP-4. Moreover, differential isotopic labeling was developed to quantify phosphorylation changes occurring during capacitation. The phosphopeptide enrichment and quantification methodology coupled to MS/MS, described here for the first time, can be employed to map and compare phosphorylation sites involved in multiple cellular processes. Although we were unable to determine the exact site of phosphorylation of VCP, we did confirm, using a cross-immunoprecipitation approach, that this protein is tyrosine phosphorylated during capacitation. Immunolocalization of VCP showed fluorescent staining in the neck of noncapacitated sperm. However, after capacitation, staining in the neck decreased, and most of the sperm showed fluorescent staining in the anterior head.After ejaculation, sperm are able to move actively but lack fertilizing competence. They acquire the ability to fertilize in the female genital tract in a time-dependent process called capacitation (1). Capacitation is accompanied by a cAMPprotein kinase-dependent increase in tyrosine phosphorylation of a subset of proteins (2, 3). Because a protein kinase cascade is involved in the regulation of the sperm fertilizing ability, it is important to characterize the proteins that undergo phosphorylation and examine how these changes relate to capacitation.Post-translational protein phosphorylation by protein kinases plays a role in many cellular processes including transduction of extracellular signals, intracellular transport, and cell cycle progression. The use of two-dimensional gel electrophoresis followed by tandem mass spectrometry (MS/MS) 1 provides a comprehensive approach to the analysis of proteins involved in cell signaling (4). Specifically, changes in tyrosine phosphorylation can be monitored using two-dimensional gel electrophoresis followed by Western blot analysis with antiphosphotyrosine (␣-PY) antibodies (5). Proteins that undergo changes in tyrosine phosphorylation during cellular pr...

Cells migrate in vivo through complex confining microenvironments, which induce significant nuclear deformation that may lead to nuclear blebbing and nuclear envelope rupture. While actomyosin contractility has been implicated in regulating nuclear envelope integrity, the exact mechanism remains unknown. Here, we argue that confinement-induced activation of RhoA/myosin-II contractility, coupled with LINC complex-dependent nuclear anchoring at the cell posterior, locally increases cytoplasmic pressure and promotes passive influx of cytoplasmic constituents into the nucleus without altering nuclear efflux. Elevated nuclear influx is accompanied by nuclear volume expansion, blebbing, and rupture, ultimately resulting in reduced cell motility. Moreover, inhibition of nuclear efflux is sufficient to increase nuclear volume and blebbing on two-dimensional surfaces, and acts synergistically with RhoA/myosin-II contractility to further augment blebbing in confinement. Cumulatively, confinement regulates nuclear size, nuclear integrity, and cell motility by perturbing nuclear flux homeostasis via a RhoA-dependent pathway.