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Characterizing the role of the Hec1 tail domain at the kinetochore-microtubule interface in human cells




Wimbish, Robert T., author
DeLuca, Jennifer, advisor
Markus, Steven, committee member
Reddy, Anireddy, committee member
Ross, Eric, committee member

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Chromosome segregation is powered by interactions between the mitotic spindle and kinetochores. Kinetochores – large, protein-rich machines built on the centromere of each sister chromatid – must bind to spindle microtubules and harness the forces from their dynamic instability to drive chromosome movement. This interaction must be robust enough to ensure chromosomes remain bound to the growing and shrinking microtubule polymers, yet must also be reversible: incorrectly oriented kinetochore-microtubule attachments can cause chromosome mis-segregation leading to aneuploidy, which can be catastrophic for the newly formed cell. Thus, cells must be able to actively regulate the strength with which kinetochores bind to spindle microtubules – such a regulatory scheme ensures that incorrect attachments can be released, and correct attachments can be preferentially stabilized. The direct linkage between kinetochores and microtubules is the highly conserved, kinetochore-anchored NDC80 complex. This complex is also an effector of attachment strength regulation; specifically, the N-terminal "tail" region of the NDC80 complex subunit Highly expressed in cancer 1 (Hec1) is a target for phosphorylation by the Aurora family of kinases, which ultimately weakens kinetochore-microtubule attachments. Here, we investigate the molecular basis for kinetochore-microtubule attachment regulation in human cells. We find that Hec1 tail phosphorylation regulates kinetochore-microtubule attachments independently of the spindle and kinetochore associated (Ska) complex, a critical factor for attachment stability, contrary to previous reports that the two pathways are functionally coupled. We additionally map the domains of the NDC80 complex required for its coordination with Ska complexes to strengthen attachments. We also find that the Hec1 tail domain is dispensable for the initial formation of kinetochore-microtubule attachments, but provide evidence it plays a role in force generation. We further interrogate this role and how phosphorylation of the tail regulates attachment formation and force generation, and find that the length requirements for these functions of the tail are different. Moreover, we demonstrate that the phospho-regulatory pathway for attachment regulation is deficient for short tails, suggesting a new model for the means by which attachments are regulated. Together these results provide novel insight into how attachments between chromosomes and the spindle are formed and regulated, and how errors in this process can lead to chromosome mis-segregation.


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chromosome segregation
cell division


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