The yeast mitotic spindle is less complex than its counterparts in larger eukaryotes and has been intensively studied using genetics, biochemistry, cell biology and ultrastructure approaches, providing an opportunity to develop an understanding of its function and regulation at a level that is not currently achievable in any other organism. The proposed studies are critical for attaining this goal. Correct establishment, function and checkpoint monitoring of kinetochore-microtubule attachments are central to mitotic fidelity. Despite much progress identifying key proteins involved in this physical attachment, and assessing their activities in vivo and in vitro, little is understood about mechanisms regulating attachments, about how microtubule-binding outer kinetochore proteins associate with the central kinetochore, or about emergent properties that result when separate subcomplexes like Dam1 and Ndc80 are together in kinetochores. Building upon discovery and analysis of the Dam1 complex, and structure studies of the Ndc80 complex, new studies will identify their binding partners, determine how Dam1 and Ndc80 complex structures relate to function, how post-translational modifications and binding partners affect function, and how ensembles of kinetochore complexes interact dynamically with microtubules. Previous discoveries that Aurora kinase regulates kinetochore attachment via Dam1 complex phosphorylation, identification of an Aurora kinase consensus site, identification of the fourth yeast Aurora kinase complex subunit, and novel implication of casein kinase 2 in inner kinetochore regulation, provide a robust foundation for studies to reveal how protein kinases regulate critical mitotic functions. Proposed studies will determine how the Aurora kinase complex is targeted to specific cellular locations, will identify its targets at each location, and will determine how phosphorylation affects activity of its targets. Molecular genetics and biochemical analysis of the fully reconstituted, four-protein Aurora kinase complex will determine how this complex is regulated. Having recently identified casein kinase 2 as a mitotic regulator of the inner kinetochore protein Ndc10 and the widely conserved Mif2 (CENP-C) linker protein, and obtained in vivo evidence for dual-regulation of these proteins by CK2 and Aurora kinase, powerful in vivo and in vitro tests of how these two kinases regulate kinetochore function will be conducted. Thanks to unique advantages of yeast, strong inroads into a full molecular dissection of mitotic spindle disassembly have already been made, and will be built upon to fully elucidate pathways and mechanisms. As cells exit mitosis, the enormously complex mitotic spindle must be disassembled rapidly, which involves taking apart stabilized microtubule bundles, disassembling protein subcomplexes, reversing post-translational modifications and destroying proteins. Because spindle disassembly mechanisms are largely unexplored, this is an extremely fertile and important area for investigation. Comprehensive genetic screens and interaction analyses will identify genes and pathways, and focused phenotypic and biochemical studies will reveal detailed mechanisms.

Public Health Relevance

These studies will increase understanding of fundamental aspects of mitotic spindle function and regulation, and since many mitotic proteins and mechanisms are evolutionarily conserved, 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 #
5R01GM047842-19
Application #
8501508
Study Section
Nuclear and Cytoplasmic Structure/Function and Dynamics Study Section (NCSD)
Program Officer
Deatherage, James F
Project Start
1992-09-30
Project End
2014-06-30
Budget Start
2013-07-01
Budget End
2014-06-30
Support Year
19
Fiscal Year
2013
Total Cost
$334,269
Indirect Cost
$105,360
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
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
Ramey, Vincent H; Wong, Amanda; Fang, Jie et al. (2011) Subunit organization in the Dam1 kinetochore complex and its ring around microtubules. Mol Biol Cell 22:4335-42
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
Nakajima, Yuko; Cormier, Anthony; Tyers, Randall G et al. (2011) Ipl1/Aurora-dependent phosphorylation of Sli15/INCENP regulates CPC-spindle interaction to ensure proper microtubule dynamics. J Cell Biol 194:137-53
Nakajima, Yuko; Tyers, Randall G; Wong, Catherine C L et al. (2009) Nbl1p: a Borealin/Dasra/CSC-1-like protein essential for Aurora/Ipl1 complex function and integrity in Saccharomyces cerevisiae. Mol Biol Cell 20:1772-84