The yeast mitotic spindle has been intensively studied using genetics, biochemistry, cell biology and ultrastructure approaches, providing an opportunity to understand its function and regulation at a level not currently achievable in any other organism. The proposed studies are critical for attaining this goal. The 10- protein Dam1 complex provides critical functions in spindle integrity and kinetochore-spindle attachment, and it is a crucial target of the budding yeast Aurora kinase. The Dam1 complex assembles into rings on microtubules that associate preferentially with the GTP-tubulin lattice, it stabilizes microtubules against disassembly, and it remains attached to the disassembling microtubule ends. These properties are ideally suited for a mechanical linkage between chromosomes and spindle microtubules. Using a recently developed real-time fluorescence assay for Dam1 ring movement and microtubule stabilization, effects of protein phosphorylation and interacting proteins on biochemical activities of the Dam1 complex will be tested. By attaching microbeads to the Dam1 complex, forces generated on the ring complex by microtubule disassembly will be measured using laser tweezers. Cryo-EM studies of Dam1 complex-coated microtubules that are already quite advanced will be used for helical reconstructions to obtain a 3D structure of the complex that can be docked onto the known structure of the microtubule surface. This analysis promises to reveal structural determinants for the function of the complex as a dynamic microtubule-binding interface, and will be extended to post-translationally modified Dam1 complex, and to the complex with associated interacting proteins. Understanding of how the Aurora kinase Ipl1 regulates mitotic spindle functions will be increased by combining bioinformatics, mass spectrometry and chemical genetics to identify additional Ipl1 target proteins. Molecular genetics will determine how phosphorylation affects target protein functions. These studies will increase understanding of fundamental aspects of mitotic spindle function and regulation, and will provide a framework for elucidating mitotic mechanisms in humans. Chromosome instability is a key contributing factor in cancer and birth defects. Therefore, understanding principles of spindle function may suggest novel strategies for prevention, detection and treatment of human diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM047842-15S1
Application #
7931575
Study Section
Nuclear Dynamics and Transport (NDT)
Program Officer
Gindhart, Joseph G
Project Start
2009-09-30
Project End
2010-12-30
Budget Start
2009-09-30
Budget End
2010-12-30
Support Year
15
Fiscal Year
2009
Total Cost
$51,326
Indirect Cost
Name
University of California Berkeley
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
124726725
City
Berkeley
State
CA
Country
United States
Zip Code
94704
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Cormier, Anthony; Drubin, David G; Barnes, Georjana (2013) Phosphorylation regulates kinase and microtubule binding activities of the budding yeast chromosomal passenger complex in vitro. J Biol Chem 288:23203-11
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Ramey, Vincent H; Wang, Hong-Wei; Nakajima, Yuko et al. (2011) The Dam1 ring binds to the E-hook of tubulin and diffuses along the microtubule. Mol Biol Cell 22:457-66
Peng, Yutian; Wong, Catherine C L; Nakajima, Yuko et al. (2011) Overlapping kinetochore targets of CK2 and Aurora B kinases in mitotic regulation. Mol Biol Cell 22:2680-9

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