Kinetochores are multiprotein organelles that orchestrate the movement of chromosomes during mitosis. Their most fundamental activity is maintaining persistent, load-bearing attachments between the chromosomes and the assembling and disassembling tips of microtubules within the mitotic spindle. This 'tip-coupling'behavior allows kinetochores to harness microtubule disassembly to produce force. It also underlies vital regulatory activities by which they ensure the accuracy of mitosis. To uncover how kinetochores perform these important functions, we are reconstituting kinetochore activities using pure components and applying new tools for manipulating and tracking individual molecules. We will use a unique combination of native kinetochore particles isolated from budding yeast, pure recombinant kinetochore subcomplexes, and state-of-the-art biophysical tools. Our in vitro approach allows long standing questions about kinetochore function to be answered in direct ways that would be impossible in living cells. Specifically, we will: (1) determine the relative contributions of the core microtubule-binding subcomplexes, Ndc80 and Dam1, to the coupling between native kinetochore particles purified from budding yeast and individual dynamic microtubule tips;(2) test whether kinetochore-microtubule coupling relies on interactions with tip-specific tubulin structures such as GTP caps, curled protofilaments, or exposed longitudinal, lateral, and luminal faces of tubulin dimers;(3) determine whether tension stabilizes kinetochore-microtubule attachments directly, independently of phosphoregulation, via a catch bond-like mechanism;(4) determine the relative contributions of two kinases, Ipl1 and Mps1, to the regulation of kinetochore-microtubule attachment stability;(5) determine whether tension suppresses phosphorylation-triggered detachment, and test candidate models for how this may occur;(6) determine whether phospho-mimicking mutations at specific sites within the Ndc80 and Dam1 subcomplexes promotes kinetochore detachment by directly weakening the attachment interface, by triggering the release of microtubule-binders from the kinetochore, or by triggering disassembly of attached microtubules. This work will help elucidate how kinetochores and other tip-couplers maintain strong yet dynamic attachments to the assembling and disassembling tips of cytoskeletal filaments, and how such attachments are regulated. Understanding the basis for these functions is essential for understanding cancer progression because chromosome loss, which occurs frequently in cancer, can result from mutations that weaken kinetochore- microtubule attachments. Promising new chemotherapeutics are being developed to target components of the mitotic machinery, and these efforts will benefit substantially from a more complete knowledge of the roles and mechanisms of specific kinetochore proteins.
During cell division, duplicated chromosomes are organized and separated by an exquisite molecular machine, the mitotic spindle ? this project will bring us closer to a complete understanding of how the spindle works. Having a mechanistic understanding of the spindle promises to revolutionize the design of chemotherapeutic drugs that target spindle components. Ultimately, it may also guide efforts to develop useful man-made nanomachines, which so far cannot match the remarkable abilities of naturally occurring protein machines.
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