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 surprisingly 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. We have shown that these complexes play important roles in kinetochore function. A number of other nucleoporins that do not associate with kinetochores have also been shown to have important mitotic roles, including Nup214, Nup98 and TPR. 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. 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. A number cancers possess chromosomal translocations that produce Nup98, Nup214 or TPR fusion proteins. While these fusions directly cause the malignant phenotype under some circumstances, the importance of the nucleoporin moieties is not understood. The fact that Nup214, Nup98 and TPR have mitotic functions raises the possibility that mitotic disregulation may contribute to development of these cancers. This idea is especially attractive for Nup98, which is promiscuous in its fusion partner utilization and which accumulates at high levels on kinetochores in cell lines expressing leukemia-associated fusions. We are currently examining the capacity of nucleoporin fusion proteins to mis-regulate mitotic spindle function and cell cycle progression. Finally, we are working to develop quantitative assays for vertebrate chromosome mis-segregation. The development of conceptually simple sectoring assays provided a powerful genetic tool in yeast to assess rates of chromosome mis-segregation and to identify mutants deficient in this process. In the absence of vital assays for vertebrate chromosome segregation, mitotic abnormalities can only be scored in live assays through the gross mis-segregation of multiple chromatids, leading to obviously unequal distribution of chromosomes to daughter cells, or through the development of highly abnormal structures, such as micronuclei or/and chromosome bridges. More subtle defects, including the kind of chromosomal instability (CIN) found in many solid tumors, can currently only be monitored through more laborious assays involving karyotype analysis or fluorescent in situ hybridization. We are working with Vladimir Larionovs laboratory, who pioneered human artificial chromosomes (HACs) as gene therapy tools for efficient and regulated expression of genes of interest, to develop practical assays for chromosome mis-segregation in vertebrate cells. These assays will straightforward, quantitative assessment of CIN under a variety of conditions. The assays will be employed particularly to monitor the impact of changes in nucleoporins, as well as in the Ran and SUMO pathways. We expect that they will also be of general utility to others in the mitosis field.
