Professor of Genetics, Genomics and Development*
*And Affiliate, Division of Cell and Developmental Biology
The mitotic spindle segregates chromosomes to daughter cells. Mistakes in chromosome segregation contribute to cancer and birth defects. Our goal is to identify the molecular mechanisms that control the fidelity of mitosis and meiosis. By employing proteomics, genetics, cellular imaging, and biochemistry, we have identified novel protein components of yeast spindles and kinetochores, and have mapped their phosphorylation sites, allowing us to perform genetic tests of the importance of these proteins and their modification by protein phosphorylation.
Structure, Function, Composition and Regulation of the Yeast Kinetochore and Mitotic Spindle. The mitotic spindle is a complex structure that must undergo a highly coordinated sequence of steps to faithfully segregate chromosomes to daughter cells. In each cell cycle the spindle must assemble, form a bipolar connection to each chromosome, segregate one copy of each chromosome to each daughter cell, and then disassemble. Using a combination of proteomics, biochemistry and genetics, we have identified many novel kinetochore and spindle proteins and sub-complexes. A particular emphasis of our work is on the kinetochore, a protein complex that joins chromosomal DNA to mitotic spindle microtubules. Although the budding yeast cell has a simple centromeric DNA sequence, the kinetochore that is assembled onto this sequence contains over 40 proteins. We would like to understand why this structure needs to contain so many subunits, how its assembly is regulated during the cell cycle, how it can associate with microtubule plus ends while allowing tubulin subunits to exchange at those ends during anaphase, and how an unattached kinetochore generates a checkpoint signal to arrest the cell cycle. We have used affinity techniques to purify kinetochore proteins and their binding partners. By examining these proteins by mass spectrometry, we have been able to identify kinetochore subcomplexes, and to build a map for kinetochore protein connectivity between the DNA and the microtubule. Moreover, phosphorylation sites within the kinetochore proteins were mapped by mass spectrometry, allowing us to genetically test the importance of these sites and to determine that the Aurora kinase Ipl1p is responsible for subset of phosphorylation events. We established that a particular protein, Dam1p, is a key target of the Aurora kinase for establishing bipolar connections between chromosomes and spindles. Molecular-genetic and biochemical studies have allowed further mapping of kinetochore subunit interactions, and determination of how these interactions are affected by proteinphosphorylation. In total, our studies are contributing to a comprehensive understanding of the molecular basis for chromosome attachment to spindles, and how this attachment is regulated.
Overlapping kinetochore targets of CK2 and Aurora B kinases in mitotic regulation. [Y. Peng, C.C. Wong, Y. Nakajima, R.G. Tyers, A.S.Sarkeshik, J. Yates 3rd, D.G. Drubin, G. Barnes (2011) Mol Biol Cell vol. 22(15):2680-9.]
Mitotic spindle disassembly occurs via distinct subprocesses driven by the Anaphase-Promoting Complex, Aurora B kinase, and kinesin-8. [ J. Woodruff, D.G. Drubin, and G. Barnes (2010) J. Cell Biol. vol. 191(4):795-808.
Dynein-driven mitotic spindle positioning restricted to anaphase by She1p inhibition of dynactin recruitment. [J.B. Woodruff, D.G. Drubin, G. Barnes (2009) Mol Biol Cell. vol. 20(13): 3003-11]
Nbl1p: a Borealin/Dasra/CSC-1-like protein essential for Aurora/Ipl1 complex function and integrity in Saccharomyces cerevisiae. [Y. Nakajima, R.G. Tyers, C.C. Wong, J.R. Yates, D.G. Drubin and G. Barnes. (2009) Mol Biol Cell. vol 20(6): 1772-84]
The Kinetochore Ring Complex Moves Processively on Depolymerizing Microtubule Ends. [S. Westermann, A. Avila-Sakar, H.-W. Wang, D.G. Drubin , E. Nogales, G. Barnes (2006) Nature vol. 440(7083): 565-570]
Formation of a dynamic kinetochore- microtubule interface through assembly of the Dam1 ring complex. [S. Westermann, A. Avila-Sakar, H.W. Wang, H. Niederstrasser, J. Wong, D.G. Drubin, E. Nogales, G. Barnes (2005) Molecular Cell. vol. 17(2):277-90]
Architecture of the budding yeast kinetochore reveals a conserved molecular core. [S. Westermann, I.M. Cheeseman, S. Anderson, J.R. Yates 3rd, D.G. Drubin, G. Barnes(2003) J Cell Biol. vol. 163(2):215-22]
Kinetochore Protein Interactions and their Regulation by the Aurora Kinase Ipl1p. [C. Shang, T.R. Hazbun, I.M. Cheeseman, J. Aranda, S Fields, D.G. Drubin, G. Barnes (2003). Mol. Biol. Cell. vol. 14(8): 3342-55]
Phospho-regulation of kinetochore-microtubule attachments by the Aurora kinase Ipl1p. [I.M. Cheeseman, S. Anderson, M. Jwa, E.M. Green, J. Kang, J.R. Yates 3rd, C.S. Chan, D.G. Drubin, G. Barnes (2002) Cell, vol. 111(2):163-172]
Photo credit: Mark Hanson at Mark Joseph Studios.
Last Updated 2011-08-12