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.

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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM079373-07
Application #
8338863
Study Section
Nuclear and Cytoplasmic Structure/Function and Dynamics Study Section (NCSD)
Program Officer
Deatherage, James F
Project Start
2006-09-29
Project End
2015-08-31
Budget Start
2012-09-01
Budget End
2013-08-31
Support Year
7
Fiscal Year
2012
Total Cost
$379,227
Indirect Cost
$107,375
Name
University of Washington
Department
Physiology
Type
Schools of Medicine
DUNS #
605799469
City
Seattle
State
WA
Country
United States
Zip Code
98195
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Jung, Seung-Ryoung; Deng, Yi; Kushmerick, Christopher et al. (2018) Minimizing ATP depletion by oxygen scavengers for single-molecule fluorescence imaging in live cells. Proc Natl Acad Sci U S A 115:E5706-E5715
LlaurĂ³, Aida; Hayashi, Hanako; Bailey, Megan E et al. (2018) The kinetoplastid kinetochore protein KKT4 is an unconventional microtubule tip-coupling protein. J Cell Biol 217:3886-3900
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Kim, Jae Ook; Zelter, Alex; Umbreit, Neil T et al. (2017) The Ndc80 complex bridges two Dam1 complex rings. Elife 6:
Asbury, Charles L (2017) Anaphase A: Disassembling Microtubules Move Chromosomes toward Spindle Poles. Biology (Basel) 6:
Kudalkar, Emily M; Deng, Yi; Davis, Trisha N et al. (2016) Coverslip Cleaning and Functionalization for Total Internal Reflection Fluorescence Microscopy. Cold Spring Harb Protoc 2016:pdb.prot085548
Kudalkar, Emily M; Davis, Trisha N; Asbury, Charles L (2016) Single-Molecule Total Internal Reflection Fluorescence Microscopy. Cold Spring Harb Protoc 2016:pdb.top077800

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