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, Ndc8O and Dami, 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, Ipli and Mpsl, to the regulation of kinetochore-microtubule attachment stability;(5) determine whether phospho-mimicking mutations at specific sites within the Ndc8O and Dami 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.

Public Health Relevance

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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Nuclear and Cytoplasmic Structure/Function and Dynamics Study Section (NCSD)
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Deatherage, James F
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University of Washington
Schools of Medicine
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Miller, Matthew P; Asbury, Charles L; Biggins, Sue (2016) A TOG Protein Confers Tension Sensitivity to Kinetochore-Microtubule Attachments. Cell 165:1428-39
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Yusko, Erik C; Asbury, Charles L (2014) Force is a signal that cells cannot ignore. Mol Biol Cell 25:3717-25
Decarreau, Justin; Driver, Jonathan; Asbury, Charles et al. (2014) Rapid measurement of mitotic spindle orientation in cultured mammalian cells. Methods Mol Biol 1136:31-40
Sarangapani, Krishna K; Duro, Eris; Deng, Yi et al. (2014) Sister kinetochores are mechanically fused during meiosis I in yeast. Science 346:248-51
Sarangapani, Krishna K; Asbury, Charles L (2014) Catch and release: how do kinetochores hook the right microtubules during mitosis? Trends Genet 30:150-9
Driver, Jonathan W; Powers, Andrew F; Sarangapani, Krishna K et al. (2014) Measuring kinetochore-microtubule interaction in vitro. Methods Enzymol 540:321-37
Tien, Jerry F; Umbreit, Neil T; Zelter, Alex et al. (2014) Kinetochore biorientation in Saccharomyces cerevisiae requires a tightly folded conformation of the Ndc80 complex. Genetics 198:1483-93
Umbreit, Neil T; Miller, Matthew P; Tien, Jerry F et al. (2014) Kinetochores require oligomerization of Dam1 complex to maintain microtubule attachments against tension and promote biorientation. Nat Commun 5:4951
Sarangapani, Krishna K; Akiyoshi, Bungo; Duggan, Nicole M et al. (2013) Phosphoregulation promotes release of kinetochores from dynamic microtubules via multiple mechanisms. Proc Natl Acad Sci U S A 110:7282-7

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