The Ran GTPase is required for many cellular functions, including nucleocytoplasmic trafficking, spindle assembly, nuclear assembly and cell cycle control. The sole nucleotide exchange factor for Ran, RCC1, binds chromatin throughout the cell cycle. The GTPase activating protein for Ran, RanGAP1, localizes to the cytosolic face of the nuclear pore complex (NPC) during interphase through association with RanBP2, a large nucleoporin. The interphase distribution of Ran regulators leads to a high concentration of Ran-GTP in nuclei, and low Ran-GTP in cytosol. The major effectors for Ran are a family of Ran-GTP binding proteins that were discovered as nuclear transport receptors. These receptors are collectively called Karyopherins; those that mediate import are called Importins, and those that mediate export are called Exportins. Karyopherins transit the NPC in a Ran- and cargo-independent fashion. Their cargo loading is governed by Ran-GTP levels: Importins bind to their cargo in the cytoplasm. Import complexes traverse the NPC and dissociate upon Ran-GTP-Importin binding. Exportins bind their cargo inside nuclei in complexes that contain Ran-GTP. After passage through the NPC, export complexes dissociate upon Ran-GTP hydrolysis. To date, two nuclear transport receptors have been shown to act as Ran effectors during mitosis, Importin-beta and Crm1. Importin-beta binds and imports cargo with classical nuclear localization sequences (cNLSs) through an adaptor subunit, Importin-alpha. In mitotic metazoan cells, Importin-alpha/beta bind and inhibit spindle assembly factor (SAFs). Elevation of diffusible Ran-GTP concentrations near mitotic chromatin releases inhibition by Importin-alpha/beta, allowing localized activation of such factors. ? SCCR studies have been particularly concerned with Ran functions at kinetochores. Kinetochores are proteinaceous structures that assemble at the centromere of each sister chromatid during mitosis, and that serve as sites of spindle microtubule (MT) attachment. The kinetochore fibers (k-fibers) that link mammalian kinetochores to spindle poles contain both MTs that are directly attached to the kinetochores at their plus ends (kMTs) and MTs that are not. Kinetochore attachment is monitored through the spindle assembly checkpoint (SAC), which prevents mitotic exit by blocking anaphase promoting complex/cyclosome (APC/C) activation until all chromosomes are attached and aligned onto the metaphase plate. The APC/C is a ubiquitin ligase that regulates the destruction of key mitotic regulatory proteins. Components of the SAC include: Mad1, Mad2, Mps1, Bub1, Bub3, BubR1, and CENP-E. ? We previously showed that elevated levels of Ran-GTP abrogate SAC-mediated mitotic arrest in Xenopus egg extracts (XEEs) and disrupt the kinetochore localization of SAC components, suggesting that the SAC is directly responsive to the overall concentration of Ran-GTP in that system. The effector for Ran in the SAC remains an unresolved issue, and this problem is a major focus of our current interests. Neither RCC1 nor Ran-GTP are required for SAC activation in response to fully unattached kinetochores in a hamster cell line with temperature-sensitive allele of the RCC1 gene (tsBN2 cells). However, experiments with tsBN2 cells indicate that Ran-GTP is required for regulation of anaphase in response to inappropriate attachments or mis-assembled k-fibers. We have also found that known mitotic effectors of Ran-GTP (Importin-alpha/beta and Crm1) are dispensible for SAC activation.? We have found that Crm1 localizes to kintochores, and that inhibition of Crm1 ternary complex formation blocks kinetochore recruitment of RanGAP1/RanBP2. Crm1 itself requires neither ternary complex assembly nor MTs for kinetochore binding. Centromeres of LMB-treated HeLa or U2OS cells were under increased tension, and these cell showed a decreased distance between their spindle poles. In LMB-treated cells, kinetochores dramatically failed to maintain discrete end-on attachments to single k-fibers and showed a resultant elevation in chromosome mis-segregation. These findings have several important implications. First, they directly link the RanGAP1/RanBP2 to correct k-fiber assembly. Second, they suggest that Ran has a kinetochore-associated effector pathway that can be clearly differentiated from Importin-beta-mediated inhibition of soluble SAFs. Third, these findings show that multiple karyopherins act as Ran effectors during mitosis; in principal, it is possible that other members of this family may also act during mitosis. The component(s) at kinetochores that is directly involved in Crm1 recruitment is a major focus of our ongoing studies. ? Finally, we have examined the function of SAC components within mitotic cells, particularly the Bub1 kinases role at the inner centromeres (IC). Protein complexes of the IC regulate sister chromatid cohesion and modulate MT attachment. These proteins include the chromosomal passenger complex (CPC), mitotic centromere-associated kinesin (MCAK), and Shugoshin (Sgo). The CPC consists of the Aurora B kinase, INCENP, Survivin, and Dasra/Borealin. Aurora B phosphorylates and inhibits the microtubule depolymerase MCAK, thereby controlling the polymerization/depolymerization state of microtubules to achieve correct end-on attachment of microtubules to kinetochores. The CPC localizes along prophase chromosomes, and concentrates at the IC in prometaphase and metaphase. The SAC component Bub1 is required for outer kinetochore assembly and for IC recruitment of Sgo. We found that Bub1 plays a central role in IC formation, acting at multiple points in this assembly pathway. First, Bub1 controls CPC localization to the IC. In the absence of Bub1, the CPC binds chromosome arms, but it does not accumulate in the IC. Although the kinase activity of Aurora B was not lost under these circumstances, CPC stability was markedly altered. Second, as in earlier reports from others, we found that Bub1 mediates xSgo recruitment to the IC. In addition, we found that Bub1 acts primarily by promoting xSgo binding to mitotic chromatin; Bub1 can accomplish this function even when it is not stably associated to mitotic chromosomes or kinetochores. Third, in contrast to chromatin binding of xSgo, Bub1 by itself is insufficient to direct xSgo to the IC in the absence of the CPC. Together, these findings suggest that Bub1 regulates localization of IC components through mechanisms that are both CPC-dependent and -independent. Remarkably, Bub1s kinase activity is essential for its roles in IC assembly, in contrast to its roles at outer kinetochores.
| Dasso, Mary; Fontoura, Beatriz M A (2018) Editorial overview: The cell nucleus: Dynamic interplay of shape and function. Curr Opin Cell Biol 52:iv-vi |
| Dasso, Mary; Fontoura, Beatriz M A (2016) Gating Immunity and Death at the Nuclear Pore Complex. Cell 166:1364-1366 |
| Dasso, Mary (2016) Kar9 Controls the Cytoplasm by Visiting the Nucleus. Dev Cell 36:360-1 |
| Zhang, Michael Shaofei; Arnaoutov, Alexei; Dasso, Mary (2014) RanBP1 governs spindle assembly by defining mitotic Ran-GTP production. Dev Cell 31:393-404 |
| Mukhopadhyay, Debaditya; Arnaoutov, Alexei; Dasso, Mary (2010) The SUMO protease SENP6 is essential for inner kinetochore assembly. J Cell Biol 188:681-92 |
| Mishra, Ram Kumar; Chakraborty, Papia; Arnaoutov, Alexei et al. (2010) The Nup107-160 complex and gamma-TuRC regulate microtubule polymerization at kinetochores. Nat Cell Biol 12:164-9 |
| Boyarchuk, Yekaterina; Salic, Adrian; Dasso, Mary et al. (2007) Bub1 is essential for assembly of the functional inner centromere. J Cell Biol 176:919-28 |
| Orjalo, Arturo V; Arnaoutov, Alexei; Shen, Zhouxin et al. (2006) The Nup107-160 nucleoporin complex is required for correct bipolar spindle assembly. Mol Biol Cell 17:3806-18 |
| Dasso, M (2006) Ran at kinetochores. Biochem Soc Trans 34:711-5 |
| Rundle, Natalie T; Nelson, Jim; Flory, Mark R et al. (2006) An ent-kaurene that inhibits mitotic chromosome movement and binds the kinetochore protein ran-binding protein 2. ACS Chem Biol 1:443-50 |
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