Trafficking between the nucleus and cytoplasm occurs through the nuclear pore complexes (NPCs). During mitosis, metazoan NPCs disassemble into approximately a dozen subunits. Two of these subunits are targeted to mitotic kinetochores: First, the RanBP2 complex associates with kinetochores in a microtubule-dependent manner. This 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 sole conjugating enzyme for the SUMO family of ubiquitin-like modifiers). Second, the nine protein vertebrate Nup107-160 complex associates to kinetochores throughout mitosis in a microtubule-independent manner. The Nup107-160 complex includes Nup160, Nup133, Nup107, Nup96, Nup85, Nup43, Nup37, Sec13, and Seh1. During telophase, Nup107-160 is targeted to chromosomes, where it acts in a critical and early fashion during NPC re-assembly. We have 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. The unattached kinetochores nucleate microtubules in a manner that is regulated by Ran GTPase;such microtubules contribute to the formation of kinetochore fibres (k-fibres), microtubule bundles connecting kinetochores to spindle poles. Our findings indicate that Nup107-160 and γ-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 Nup107-160gamma-TuRC complexes nucleated microtubule formation in assays using purified tubulin. Although Ran did not regulate microtubule nucleation by gamma-TuRC alone, Nup107-160gamma-TuRC complexes required RanGTP for microtubule nucleation. Collectively, our observations show that Nup107-160 promotes spindle assembly through RanGTP-regulated nucleation of microtubules by gamma-TuRC at kinetochores, and reveal a novel relationship between nucleoporins and the microtubule cytoskeleton. Our future studies will focus on a number of issues, including the component(s) at kinetochores that is directly involved in Crm1 recruitment, the relationship between the Nup107-160 and RanBP2 complexes during mitosis, and how they together regulate the attachment of microtubules to kinteochores and the spindle assembly checkpoint. We are also examining the mitotic localization and function of other NPC components.
