Chromosome segregation during mitosis requires a large proteinaceous structure termed the kinetochore to generate attachments between chromosomal DNA and spindle microtubule polymers. The kinetochore is composed of more than 80 different proteins which function together to direct kinetochore assembly, generate dynamic connections with spindle microtubules, and regulate chromosome segregation. Our lab is interested in understanding the molecular basis of kinetochore function in human cells. We use parallel biochemical and cell biological approaches to examine kinetochore composition, structure, organization, regulation, and how kinetochore proteins function to achieve proper chromosome segregation.
Human tissue culture cell in mitosis shows microtubules in green, DNA in blue,and kinetochores in red.
We have developed an affinity tagging strategy (the Localization and Affinity Purification – LAP Tag) that facilitates both the cell biological and biochemical analysis of proteins in human tissue culture cells. Using this tag, we are conducting proteomics of human kinetochores to identify new human kinetochore proteins, and to define the organization of kinetochore components in biochemically defined sub-complexes.
In addition to identifying new proteins, we are using the mass spectrometry-based analysis of kinetochore proteins to map post-translational modifications such as phosphorylation. We are identifying an extensive collection of phosphorylation sites present in human kinetochore proteins. We are currently working to determine which protein kinases are responsible for targeting these different sites, and to examine the physiological consequences of phosphorylation on protein activities in vitro, and chromosome segregation and kinetochore function in cells.
Kinetochore Structure and Function
To examine the structure and activities of kinetochore protein complexes, we are biochemically reconstituting kinetochore proteins using recombinant expression strategies. We have recently reconstituted a nine subunit protein group termed the KNL-1/Mis12 Complex/Ndc80 Complex (KMN) network. Using in vitro assays, we have examined the organization and assembly of this network, and demonstrated that the KMN network is able to bind to microtubules directly. Based on the observed microtubule binding properties, and the work that we and others have done on this network in vivo, the KMN network is a core component of the interface with microtubules. We have also demonstrated that the binding of the Ndc80 complex to microtubules is sensitive to phosphorylation from Aurora B kinase, a key regulator of kinetochore-microtubule attachments. We are currently working to dissect the structure and function of the KMN network, as well as apply this strategy to other kinetochore protein complexes that we have isolated.
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Hori, T., M. Amano, A. Suzuki, C. B. Backer, J. P. I. Welburn, Y. Dong, B. F. McEwen, E. Suzuki, J. R. Yates III, K. Okawa, I. M. Cheeseman, and T. Fukagawa (2008). “The CCAN makes multiple contacts with centromeric DNA and provides distinct pathways to the outer kinetochore.” Cell 135: 1039-1052.
Cheeseman, I. M., T. Hori, T. Fukagawa, and A. Desai (2008). “hKNL1 and the CENP-H/I/K Complex Coordinately Direct Kinetochore Assembly in Vertebrates”. Molecular Biology of the Cell 19: 587-594.
Cheeseman, I. M. and A. Desai (2008). “Molecular Architecture of the Kinetochore-Microtubule Interface”. Nature Reviews Molecular Cell Biology 9: 33-46.
Cheeseman, I. M., J. S. Chappie, E. M. Wilson-Kubalek, and A. Desai (2006). “The Conserved KMN Network Constitutes the Core Microtubule Binding Site of the Kinetochore”. Cell 127 (5): 983-997.
Cheeseman, I. M., S. Niessen, S. Anderson, F. Hyndman, J. R. Yates, K. Oegema, and A. Desai (2004). “A Conserved Protein Network Controls Assembly of the Outer Kinetochore and Its Ability to Sustain Tension.” Genes & Development 18 (18): 2255-2268.
Cheeseman, I. M., S. Anderson, M. Jwa, E. Green, J. Kang, J. R. Yates, C. S. M. Chan, D. G. Drubin, and G. Barnes (2002). “Phospho-regulation of Kinetochore-Microtubule Attachments by the Aurora Kinase Ipl1p.” Cell 111 (2): 163-172.