The long-term objective of this research is to use cell biological and biochemical techniques to understand the molecular nature of the kinetochore-microtubule (MT) interface in vertebrate cells. For chromosomes to properly segregate during mitosis, they must firmly attach to dynamically growing and shortening MT plus- ends. They do so via the kinetochore, a large protein assemblage built at the primary constriction of mitotic chromosomes. This linkage is likely complicated, as it must be robust to resist the forces of chromosome bi- orientation, yet flexible to allow for fluid growth and shortening of bound MT plus-ends. Kinetochores must also regulate the strength of this attachment, since incorrectly attached MTs must be released, and those that are correctly attached must be stabilized. The kinetochore-associated NDC80 complex is required for generating stable kinetochore-MT attachments in eukaryotic cells, but how this complex builds and regulates binding sites for the plus-ends of spindle MTs remains one of the most important unanswered questions in the mitosis field. This proposal is designed to answer the following questions: What domains of the NDC80 complex make up the direct points of contact with MT plus-ends? Are the complexes tethered together in a "sleeve" for the MT plus-ends to insert into? If so, how are the complexes tethered together? Do weakly-associated NDC80 complexes diffuse along the MT lattice to facilitate chromosome congression? Or, alternatively, are the points of contact between kinetochores and MTs made up of high affinity binding interactions that require continual release and re-binding to drive chromosome movement? Is kinetochore-MT binding strength regulated through phosphorylation of the NDC80 complex by Aurora B kinase? What phosphatase counter-acts the kinase activity to ensure kinetochore-MT stabilization? These questions will be answered using the following approaches: First, a gene silence/rescue strategy for NDC80 complex components will be developed in PtK1 cells to unambiguously assess kinetochore-MT attachment phenotypes in cells expressing mutant NDC80 complexes. Second, biochemical and biophysical experiments using NDC80 mutants will be carried out to understand mechanistically how NDC80 complexes bind to and translocate along MTs, and which features of the complex are responsible for physically coupling plus-end MT dynamics to force production for chromosome movement. Third, protein-protein interactions will be mapped at the kinetochore-MT interface for the first time in vivo through the development of kinetochore-specific fluorescence interaction assays. These studies will provide answers to a critical set of unresolved questions in the mitosis field, and the developed techniques will be applicable to further study of mitotic proteins and processes. Relevance: Progression through mitosis with incorrect kinetochore-MT attachments is a major cause of aneuploidy, which has been linked to the initiation and progression of human tumors and also to the formation of birth defects. Thus, understanding how cells generate and regulate kinetochore-MT attachments is of critical importance to human health.

Public Health Relevance

During mitosis, chromosomes must segregate correctly in order to prevent the formation of aneuploid cells, which contain an incorrect number of chromosomes. This is critical for human health, as aneuploidy is well-known for causing birth defects and has been implicated as a causative factor in the initiation and progression of tumors. Understanding the mechanisms that cells use to correctly divide their chromosomes equally into two daughter cells is essential to understand the pathways leading to the emergence of aneuploid cells.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM088371-04
Application #
8447488
Study Section
Nuclear and Cytoplasmic Structure/Function and Dynamics Study Section (NCSD)
Program Officer
Deatherage, James F
Project Start
2010-04-01
Project End
2015-03-31
Budget Start
2013-04-01
Budget End
2014-03-31
Support Year
4
Fiscal Year
2013
Total Cost
$274,798
Indirect Cost
$85,071
Name
Colorado State University-Fort Collins
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
785979618
City
Fort Collins
State
CO
Country
United States
Zip Code
80523
Herman, Jacob A; Toledo, Chad M; Olson, James M et al. (2015) Molecular pathways: regulation and targeting of kinetochore-microtubule attachment in cancer. Clin Cancer Res 21:233-9
Zaytsev, Anatoly V; Sundin, Lynsie J R; DeLuca, Keith F et al. (2014) Accurate phosphoregulation of kinetochore-microtubule affinity requires unconstrained molecular interactions. J Cell Biol 206:45-59
Caldas, Gina V; DeLuca, Jennifer G (2014) KNL1: bringing order to the kinetochore. Chromosoma 123:169-81
Toledo, Chad M; Herman, Jacob A; Olsen, Jonathan B et al. (2014) BuGZ is required for Bub3 stability, Bub1 kinetochore function, and chromosome alignment. Dev Cell 28:282-94
Suzuki, Aussie; Badger, Benjamin L; Wan, Xiaohu et al. (2014) The architecture of CCAN proteins creates a structural integrity to resist spindle forces and achieve proper Intrakinetochore stretch. Dev Cell 30:717-30
Earnshaw, W C; Allshire, R C; Black, B E et al. (2013) Esperanto for histones: CENP-A, not CenH3, is the centromeric histone H3 variant. Chromosome Res 21:101-6
Kalashnikova, Anna A; Winkler, Duane D; McBryant, Steven J et al. (2013) Linker histone H1.0 interacts with an extensive network of proteins found in the nucleolus. Nucleic Acids Res 41:4026-35
Caldas, Gina V; DeLuca, Keith F; DeLuca, Jennifer G (2013) KNL1 facilitates phosphorylation of outer kinetochore proteins by promoting Aurora B kinase activity. J Cell Biol 203:957-69
DeLuca, Jennifer G; Musacchio, Andrea (2012) Structural organization of the kinetochore-microtubule interface. Curr Opin Cell Biol 24:48-56
DeLuca, Keith F; Lens, Susanne M A; DeLuca, Jennifer G (2011) Temporal changes in Hec1 phosphorylation control kinetochore-microtubule attachment stability during mitosis. J Cell Sci 124:622-34

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