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 NPC: Many 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 XEE caused a SAC-dependent delay in anaphase onset, suggesting that endogenous p31comet is important for mitotic timing in this system. 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. Regulation of deoxyribonucleotide production during mitosis: IRBIT (inositol-1,4,5- trisphosphate (IP3) receptor-binding protein) forms a dATP-dependent complex with ribonucleotide reductase (RNR) that is regulated by the Ran GTPase pathway during mitosis. RNR provides deoxynucleotide triphosphates (dNTPs) for genomic and mitochondrial DNA replication and repair. Uncontrolled RNR activity has been associated with malignant transformation and tumor cell growth. RNR is subject to allosteric regulatory mechanisms in all eukaryotes, as well as to control by small protein inhibitors in budding and fission yeast. The key role of RNR in DNA synthesis has made it a target for both anticancer and antiviral therapy. We were thus interested to understand the functional consequences of the IRBIT-RNR interaction. We found that IRBIT is an allosteric inhibitor of RNR, which binds the dATP-bound form of RNR and suppresses its activity. HeLa cells showed imbalanced pools of dNTPs after IRBIT depletion. We found that this effect was most pronounced during the mitotic phase of the cell cycle, but that dNTP pools were less sensitive during G1 phase. Consistent with these findings, we observed that IRBIT bound to RNR more strongly during mitosis than during G1 phase. Live imaging of tissue culture cells demonstrated that IRBIT depletion caused an acceleration of the cell cycle, as well as much greater variation in between individual cells in overall cell cycle duration. IRBIT loss was associated with changes in DNA replication patterns and disruption of genomic stability. 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 AlphoidtetO 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. Missegregation 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. RanBP1 dynamically controls RCC1 activity during mitosis: The Ran pathway organizes many functions during mitosis with respect to chromosomes. After NEB, Ran-GTP is concentrated near mitotic chromatin, and Ran distal to chromosomes is mostly GDP bound. This Ran-GTP gradient guides mitotic spindle assembly. We have examined how Ran-GTP gradients are formed and dynamically regulated. Notably, the association of RCC1, Rans guanine nucleotide exchange factor (GEF), to chromatin changes dramatically during mitosis, with large increases in the amount of chromatin-bound RCC1 shortly after the metaphase-anaphase transition. Moreover, the fact that elevated RCC1 levels disrupt kinetochore structures and SAC signaling suggests that the dynamics of RCC1 has important functional consequences. RanBP1 is a Ran-GTP-binding protein that forms a stable heterotrimeric complex with RCC1 and Ran (RRR complex), inhibiting RCC1s RanGEF activity. We have shown that the RRR complex forms readily in M-phase XEEs. RCC1 binding to chromatin and RRR complex assembly were mutually exclusive, so that promoting RRR complex formation through the addition of recombinant xRanBP1 sequestered RCC1 away from chromatin. Moreover, RRR complex assembly inhibited RCC1s RanGEF activity in CSF-XEE, in agreement with earlier in vitro observations. Together, these findings suggest that the RRR complex plays a pivotal mitotic role in determining the partitioning of RCC1 between its active chromatin-bound and inactive soluble states, thereby setting both the location and magnitude of Ran-GTP production during mitosis. Furthermore, we observed that RanBP1 is phosphorylated during anaphase in cycling XEE and that this modification disrupts RRR complex assembly. Our data suggest that modification of RanBP1 drives changes in RCC1 binding to chromatin and thereby indirectly alters the rate of Ran-GTP production in anaphase. We speculate that this mechanism may not only cause dramatic changes in Ran-GTP levels important for mitotic exit and NE reassembly but also help to re-establish correct configuration of the Ran pathway during the subsequent interphase.
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