Trafficking between the nucleus and cytoplasm occurs through the nuclear pore complexes (NPCs). Kinetochores are proteinaceous structures that assemble at the centromere of each sister chromatid during mitosis, and that serve as sites of spindle microtubule attachment. The relationship between mitotic kinetochores and the NPC is both surprising intimate and poorly understood. NPCs consists of around thirty proteins, called nucleoporins. During interphase, a number of kinetochore proteins also stably bind to NPCs (e.g., Mad1, Mad2, Mps1). During mitosis, metazoan NPCs disassemble, and at least a third of nucleoporins associate with kinetochores, including the RanBP2 complex complex and the Nup107-160 complex. The RanBP2 complex consists of RanBP2 (a large nucleoporin that is also known as Nup358), SUMO-1-conjugated RanGAP1 (the activating protein for the Ran GTPase) and Ubc9 (the conjugating enzyme for the SUMO family of ubiquitin-like modifiers). This complex associates with kinetochores in a microtubule-dependent manner. Disruption of RanBP2 association to kinetochores causes defective mitotic spindle assembly. During interphase, a small fraction of endogenous RanBP2 interacts with the plus-ends of microtubules through its N-terminal region (BPN). Cells overexpressing the isolated BPN domain displayed dramatic alterations in their microtubule organization, including microtubule bundling and hyper-stabilization. Cells ectopically expressing of BPN or full-length RanBP2 exhibited significantly higher levels of acetylated microtubules that were resistant to nocodazole, a microtubule depolymerizing agent. RNAi mediated depletion of RanBP2 affected microtubule polarization during directed cell migration, further suggesting an in vivo role of RanBP2 in interphase microtubule organization. Our current studies on this complex focus on interacting proteins that may be essential for the function of the RanBP2 complex in mitosis. The nine protein vertebrate Nup107-160 complex includes Nup160, Nup133, Nup107, Nup96, Nup85, Nup43, Nup37, Sec13, and Seh1. The Nup107-160 complex associates to kinetochores throughout mitosis in a microtubule-independent manner. During telophase, Nup107-160 is targeted to chromosomes, where it acts in a critical and early fashion during NPC re-assembly. Unattached kinetochores nucleate microtubules in a manner that requires the small GTPase Ran;such microtubules promote assembly of kinetochore fibers (k-fibers) that connect kinetochores to spindle poles. We found that Nup107-160 interacts with the gamma-tubulin ring complex (gamma-TuRC), an essential and conserved microtubule nucleator, and recruits gamma-TuRC to unattached kinetochores. Nup107-160 and gamma-TuRC act cooperatively to promote spindle assembly through microtubule nucleation at kinetochores: HeLa cells lacking Nup107-160 or gamma-TuRC were profoundly deficient in kinetochore-associated microtubule nucleation. Moreover, co-precipitated Nup107-160/gamma-TuRC complexes nucleated microtubule formation in assays using purified tubulin. Although Ran did not regulate microtubule nucleation by gamma-TuRC alone, Nup107-160/gamma-TuRC complexes required Ran-GTP for microtubule nucleation. Collectively, our observations show that Nup107-160 promotes spindle assembly through Ran-GTP-regulated nucleation of microtubules by gamma-TuRC at kinetochores, and reveal a novel relationship between nucleoporins and the microtubule cytoskeleton. Notably, kinetochore recruitment of the RanBP2 complex is coupled with kinetochore-MT attachment and inhibits nucleation at kinetochores, possibly through suppression of Nup107-160/gamma-TuRC complexes. We are currently investigating the mechanism through which Ran regulates microtubule nucleation of gamma-TuRC associated with Nup107-160 at kinetochores.
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