The transmission of genetic material in each cell division requires its accurate duplication and distribution to the daughter cells. Errors in this process lead to aneuploidy, which is implicated in oncogenesis, disability and cell death. The chromosomes are segregated into two equivalent parts by a microtubule-based molecular machine, the mitotic spindle. The spindle is bipolar with each pole carrying an exact complement of chromosomes to each daughter cell. Spindle morphogenesis requires spatially controlled microtubule nucleation. The small gamma-tubulin complex is required for nucleation but is surprisingly inactive in isolated form. Our recent structural advances make a strong prediction for how the complex could be activated, which we will test by a mutational analysis. Moreover we will identify components or modifications of the complex that activate and regulate nucleation. Chromosomes attach to microtubules via kinetochores, multiprotein organelles that span from the DNA to the microtubule. This connection is carefully orchestrated and controls both the movement and organization of chromosomes to ensure proper segregation. Kinetochores perform the surprising feat of maintaining strong persistent attachments to microtubules even during the addition and removal of thousand of tubulin subunits. We will continue our reconstitution of the kinetochore from component parts to understand the molecular mechanism and regulation underlying this attachment.

Public Health Relevance

The transmission of genetic material in each cell division requires its accurate duplication and distribution to the daughter cells. Errors in this process lead to aneuploidy, which is implicated in oncogenesis, disability and cell death.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM040506-25
Application #
8610318
Study Section
Nuclear and Cytoplasmic Structure/Function and Dynamics Study Section (NCSD)
Program Officer
Deatherage, James F
Project Start
1988-07-01
Project End
2015-01-31
Budget Start
2014-02-01
Budget End
2015-01-31
Support Year
25
Fiscal Year
2014
Total Cost
$466,282
Indirect Cost
$160,295
Name
University of Washington
Department
Biochemistry
Type
Schools of Medicine
DUNS #
605799469
City
Seattle
State
WA
Country
United States
Zip Code
98195
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
Asbury, Charles L; Tien, Jerry F; Davis, Trisha N (2011) Kinetochores' gripping feat: conformational wave or biased diffusion? Trends Cell Biol 21:38-46
Graczyk, Beth; Davis, Trisha N (2011) An assay to measure the affinity of proteins for microtubules by quantitative fluorescent microscopy. Anal Biochem 410:313-5
Tien, Jerry F; Umbreit, Neil T; Gestaut, Daniel R et al. (2010) Cooperation of the Dam1 and Ndc80 kinetochore complexes enhances microtubule coupling and is regulated by aurora B. J Cell Biol 189:713-23
Shimogawa, Michelle M; Wargacki, Megan M; Muller, Eric G et al. (2010) Laterally attached kinetochores recruit the checkpoint protein Bub1, but satisfy the spindle checkpoint. Cell Cycle 9:3619-28
Franck, Andrew D; Powers, Andrew F; Gestaut, Daniel R et al. (2010) Direct physical study of kinetochore-microtubule interactions by reconstitution and interrogation with an optical force clamp. Methods 51:242-50
Riffle, Michael; Davis, Trisha N (2010) The Yeast Resource Center Public Image Repository: A large database of fluorescence microscopy images. BMC Bioinformatics 11:263
Kollman, Justin M; Polka, Jessica K; Zelter, Alex et al. (2010) Microtubule nucleating gamma-TuSC assembles structures with 13-fold microtubule-like symmetry. Nature 466:879-82
Fonslow, Bryan R; Kang, Seong A; Gestaut, Daniel R et al. (2010) Native capillary isoelectric focusing for the separation of protein complex isoforms and subcomplexes. Anal Chem 82:6643-51
Wargacki, Megan M; Tay, Jessica C; Muller, Eric G et al. (2010) Kip3, the yeast kinesin-8, is required for clustering of kinetochores at metaphase. Cell Cycle 9:2581-8

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