Molecular mechanisms controlling kinetochore-microtubule attachments during mitosis

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Project Summary: Mitotic cells assemble two key cellular structures to segregate the chromosomes equally between the two daughter cells - the mitotic spindle and the kinetochores. Kinetochores are multi-protein complexes that form at the centromeres of the chromosomes during mitosis and serve as attachment sites for microtubules [MT(s)] of the mitotic spindle. The kinetochore-microtubule (kMT) interface generates force that drives chromosome alignment and segregation. The current focus of the Varma lab is on understanding the molecular mechanisms involved in kMT attachments and their contribution to accurate chromosome segregation. During early mitotic prometaphase, kinetochores initially attach to the MT lattice laterally. These lateral attachments are subsequently converted into end-on attachments when sister kinetochores become stably attached to the plus-ends of spindle MTs in metaphase. Studies have shown that the initial capture and lateral sliding of kinetochores on MTs is driven by dynein, a minus-end-directed motor. The end-on kinetochore- microtubule (kMT) attachment formation and its stabilization is mediated by the MT-binding kinetochore complex, Ndc80. Our 1st major goal is to determine how the kinetochore complexes required for these two alternate modes of MT attachment coordinate to produce dynamic kMT attachments required for proper chromosome alignment. Our recent work has provided evidence for an antagonistic relationship between dynein and the Ndc80 complexes in humans during mitosis. Our unpublished results suggest that dynein and the Ndc80 complex synergize for efficient chromosome capture and for stabilizing kMT attachments during metaphase, but the mechanism for this coordination is unclear. The centromere-distal region of the Ndc80 complex at the N-terminal domain of the Hec1 subunit has been identified as the MT-binding site required to stabilize kMT attachments. Phosphorylation of this region by Aurora B kinase negatively regulates the strength of kMT attachments. Our work has discovered that in addition to the N-terminal domain, the more internal loop domain has a major role in attachment. The attachment requires the loop domain-mediated kinetochore recruitment of the replication licensing protein Cdt1, which we find is a novel MT-binding protein at kinetochores. Our work also demonstrates that the binding of Cdt1 to MTs is negatively regulated by Aurora B. Our unpublished studies demonstrate that Cdt1 synergizes with another MT-associated protein (MAP) at kinetochores, the Ska complex that has been shown to promote efficient binding of the Ndc80 complex to kMTs. Our 2nd major goal is to determine how different kinetochore MAPs mediate robust interaction between the Ndc80 complex and MTs for the stabilization of kMT attachments during metaphase to drive accurate chromosome segregation. In the long term, our lab aims to identify novel mechanisms controlling kMT attachments and the pathways that regulate this process, while also establishing novel model systems and approaches to study these processes.