Kinetochores are proteinaceous structures that act as both chromosome attachment sites for spindle microtubules and as signaling platforms that control mitotic progression. Prior to achieving correct attachment to spindle microtubules, kinetochores activate the spindle assembly checkpoint (SAC), an inhibitory pathway that delays the onset of anaphase by preventing activation of the Anaphase promoting complex/cyclosome (APC/C) as a ubiquitin ligase. There is a profound but poorly understood relationship between kinetochores and the Nuclear Pore Complex (NPC): Many NPC components (nucleoporins) associate with kinetochores during mitosis in metazoan cells, while kinetochore proteins frequently reside at NPCs during interphase. Nucleoporins control multiple facets of mitotic function, including spindle assembly, microtubule dynamics, the SAC and MT-kinetochore interactions. They may also promote successful completion of mitosis by coordinating these functions. Notably, chromosomal translocations generating nucleoporin fusion proteins, changes in nucleoporin expression levels and other mutations have been linked to human cancers, and, in some cases, directly implicated in the development of aneuploidy. Our studies have demonstrated novel biochemical mechanisms through which nucleoporins act within mitosis. Recent studies include: Activation of p31comet by phosphorylation in anaphase: To understand SAC regulation, we have investigated p31comet, a protein of higher eukaryotes that plays an important role in SAC silencing after attachment has been achieved. We found that p31comet depletion from Xenopus egg extracts (XEE) caused a SAC-dependent delay in anaphase onset, suggesting that endogenous p31comet is important for mitotic timing. p31comet was mitotically phosphorylated in XEE, and a phosphomimetic p31comet mutant showed increased activity in dissociation of soluble MCC and particularly in disruption of kinetochore association of SAC components. We tested whether a number of well-established mitotic kinases, but observed that they did not efficiently phosphorylate p31comet. As an alternative candidate, we examined IKK-beta (Inhibitor of nuclear factor kappa-B kinase-beta). We found that IKK-beta was an efficient p31comet kinase, and that depletion or inhibition of IKK-beta delayed mitotic exit of XEEs. Together, our results suggest that p31comet contributes to the timing of anaphase onset in XEE through antagonism of the SAC and that IKK-beta modifies p31comet to enhance its activity. Future experiments will extend this observation to somatic cell systems, as well as examine the control of p31comet phosphorylation during mitotic exit. Development of novel assays for chromosome instability (CIN) that utilize human artificial chromosomes (HACs): HACs have been extensively developed as potential vectors for gene therapy, and it has previously been shown that their segregation relies on the same machinery that mediates endogenous chromosome segregation. To develop a live cell assay for CIN, we reengineered the Alphoid-tetO HAC to express a fluorescent marker that is cyclically degraded during each mitosis, enhanced green fluorescent protein (eGFP) fused to the destruction box (DB) domain of the APC/C substrate hSecurin. Mis-segregation of the HAC during any mitosis results in the production of daughter cells that lack the HAC and that therefore remain non-fluorescent during the subsequent cell cycle. The reengineered HAC also expresses the tetracycline repressor protein fused to monomeric cherry fluorescent protein (tetR-mCherry), which binds to tetO arrays within the HAC itself, giving us an independent marker for assessment of HAC segregation. We have extensively characterized the behavior and segregation of the HAC within a human U2OS-based cell line (U2OS-Phoenix). We found that this assay provides a useful, quantitative measurement of CIN. We further assayed CIN levels in U2OS-Phoenix cells treated with well-studied agents that target microtubule dynamics or the SAC at sub-lethal concentrations that did not cause mitotic arrest. Our results show important quantitative differences between these compounds, and demonstrate that our assay not only differentiates between these drugs but also provides the capacity to detect CIN directly without the necessity to score for morphological changes. We are currently working to develop additional HAC-based assays for CIN, and to employ those assays for high-throughput screening to identify CIN regulators. Analysis of cell lines with conditionally unstable nucleoporin proteins: Understanding the mitotic roles of nucleoporins is complicated by the fact that they are essential during both interphase and mitosis. While mutation or RNAi-mediated depletion of nucleoporins cause mitotic defects in many organisms, it is hard to fully exclude the possibility that these phenotypes result from aberrant nuclear trafficking during the preceding interphase. To circumvent this problem, we are using the CRISPR/Cas9 system for biallelic targeting of the roughly 30 genes that encoding all major nucleoporins, introducing an auxin-inducible degron (AID) tag and a fluorescent tag. For each of these genes, we will analyze nucleoporin localization and stability in the absence and presence of auxin, as well as the trafficking of import and export cargo through the NPC and other cellular functions. Upon validating the cell lines, they will be used to address a number of important questions through the selective depletion of individual nucleoporins at specified points in the cell cycle, including the contribution of those nucleoporins to the architecture of the NPC and of kinetochores, as well as their function in spindle assembly and the SAC. We anticipate that these studies will provide important insights into the spatiotemporal roles of the nuclear trafficking machinery within mitosis, how these activities are controlled during mitotic progression and how the disregulation of these components could contribute to chromosome mis-segregation and the development of aneuploidy.