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 (MT)-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 MT-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 and it acts in a critical and early fashion during NPC re-assembly. ? Our earlier studies suggested that mitotic RanBP2 complex targeting occurs through the RanBP2 protein, and that its recruitment is both regulated by and essential for correct MT-kinetochore attachment. We further found that the Crm1 nuclear export receptor localizes to kinetochores and promotes the binding of the RanBP2 complex. During the past year, we examined the mitotic role of Nup107-160 complex in collaboration with Douglass Forbes (UCSD), using HeLa tissue culture cells and Xenopus egg extracts (XEEs). In Hela cells, this complex localized not only kinetochores but also to prometaphase spindle poles and proximal spindle fibers, reminiscent of the localization of some spindle assembly checkpoint (SAC) components. Expanded crescents of the Nup107-160 complex formed around unattached kinetochores, similar to the accumulation observed of dynamic outer kinetochore proteins. In mitotic XEEs, the Nup107-160 complex localized throughout reconstituted spindles. When it was depleted from XEEs, the SAC remained intact and responsive to Ran-GTP concentration, but spindle assembly was defective. MT nucleation around sperm centrosomes appeared normal, but the MTs quickly disassembled, leaving largely unattached sperm chromatin. Notably, Ran-GTP could still promote assembly of MT asters in Nup107-160 depleted extracts, indicating that Nup107-160 is not essential for soluble SAFs activation. Together, our findings showed that the Nup107-160 complex is dynamic in mitosis, and that it promotes spindle assembly in a context that is distinct from intact interphase NPCs. ? 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.
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