Accurate chromosome segregation is important for human health because its failure can lead to congenital malformations and is part of a cancer's progression to malignancy. A cell's ability to achieve mitotic and meiotic precision depends on kinetochore-microtubule (MT) interactions, which are the major source for the forces that organize and move chromosomes in preparation for cell division. Many proteins are involved, especially in the connections with MT plus ends that form as chromosomes become bi-oriented, but how these molecules do their jobs is currently unknown. Some of the relevant proteins are motor enzymes, some are MT binding proteins without motor activity, and some are regulatory enzymes that adjust the strength of MT binding, helping to eliminate improper connections. Experiments in yeasts have shown that chromosome movement depends less on kinetochore motors than non-motor, MT-binding proteins, which emphasizes the importance of these non-enzymatic links in mitosis for all kinds of cells. Images of mitotic kinetochores obtained by electron tomography suggest that at least some of the links between kinetochores and MT ends are made directly with the bent protofilaments that flare out at the ends of these dynamic polymers. Here we propose to study such links in human cells by pursuing promising leads discovered during our previous period of funding. The N-terminal moiety of centromere protein F (CENP-F) binds preferentially to oligomers of tubulin that are curled, whereas the C-terminal MT-binding site of this protein prefers MT walls. Both these interactions are likely to be important for mitosis because the connections between kinetochores and MT ends are the sites at which force is exerted on chromosomes and where the addition and loss of tubulin occurs during chromosome motion to and from the spindle poles. We will analyze the interactions of MTs with recombinant polypeptides generated from CENP-F cDNAs, using biochemical, cell biological and biophysical methods. The resulting data will clarify how MT plus-end dynamics can exert force on this MT-kinetochore coupler and show how CENP-F contributes to precise chromosome movement. MT binding sites on CENP-F will be mapped by protein cross- linking, proteolysis, and mass spectrometry. Amino acids important to these sites will be mutated, allowing us to measure their contribution to MT-CENP-F interactions in vitro. Both truncations of CENP-F and the point mutants identified as significant for MT binding will then be examined in vivo, using a recombinase-mediated cassette exchange strategy to generate stable mutant human cell lines. Thanks to RNA interference, either dominant or recessive mutants can be characterized for their effects on mitotic chromosome behavior. The biophysics of CENP-F interaction with dynamic MTs in vitro will let us characterize these interactions and their abilityto transduce significant force from MT shortening and growth. Together these results will elucidate the role(s) of CENP-F in the accurate segregation of human chromosomes and tell us whether this MT-kinetochore coupler might be a suitable target for novel drugs to arrest mitosis and inhibit unwanted cell growth.
The mitotic spindle is the cellular machine that organizes and segregates the chromosomes as a cell prepares to divide;proper spindle function is essential for normal human development, adult tissue maintenance, and wound healing, but its mechanisms are not yet fully understood. To improve our understanding of mitosis we will study the protein CENP-F, which helps to bind human chromosomes to the fibers of the mitotic spindle. We will use biochemical, cell-biological, and biophysical methods to characterize the two distinct spindle-fiber binding sites on CENP-F and learn how they contribute to the functionally significant ways in which the spindle segregates chromosomes.
| McIntosh, J Richard; O'Toole, Eileen; Morgan, Garry et al. (2018) Microtubules grow by the addition of bent guanosine triphosphate tubulin to the tips of curved protofilaments. J Cell Biol 217:2691-2708 |
| Blackwell, Robert; Edelmaier, Christopher; Sweezy-Schindler, Oliver et al. (2017) Physical determinants of bipolar mitotic spindle assembly and stability in fission yeast. Sci Adv 3:e1601603 |
| Morphew, Mary K; Giddings Jr, Thomas H; McIntosh, J Richard (2017) Immunolocalization of Proteins in Fission Yeast by Electron Microscopy. Cold Spring Harb Protoc 2017: |
| Blackwell, Robert; Sweezy-Schindler, Oliver; Edelmaier, Christopher et al. (2017) Contributions of Microtubule Dynamic Instability and Rotational Diffusion to Kinetochore Capture. Biophys J 112:552-563 |
| Morphew, Mary K; Giddings Jr, Thomas H; McIntosh, J Richard (2017) Cryoelectron Microscopy of Fission Yeast. Cold Spring Harb Protoc 2017: |
| McIntosh, J Richard; Morphew, Mary K; Giddings Jr, Thomas H (2017) Electron Microscopy of Fission Yeast. Cold Spring Harb Protoc 2017: |
| Zhao, Xiaowei; Schwartz, Cindi L; Pierson, Jason et al. (2017) Three-Dimensional Structure of the Ultraoligotrophic Marine Bacterium ""Candidatus Pelagibacter ubique"". Appl Environ Microbiol 83: |
| Giddings Jr, Thomas H; Morphew, Mary K; McIntosh, J Richard (2017) Preparing Fission Yeast for Electron Microscopy. Cold Spring Harb Protoc 2017: |
| McIntosh, J Richard (2016) Mitosis. Cold Spring Harb Perspect Biol 8: |
| McIntosh, J Richard; Hays, Thomas (2016) A Brief History of Research on Mitotic Mechanisms. Biology (Basel) 5: |
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