The accurate segregation of chromosomes at mitosis is essential for proper cell division and for the development of all healthy animals and plants. Proper mitosis, in turn, depends on correct interactions between kinetochores and spindle microtubules. We have developed cell biological and biophysical methods to study the interactions between isolated kinetochore proteins and dynamic microtubules in vitro under conditions that mimic several aspects of normal chromosome-spindle fiber attachment in cells. With these and novel assays that are under development we will critically examine the mechanisms of action of molecular "couplers", assemblies of kinetochore proteins that can harness dynamic microtubule ends to kinetochores, allowing the transduction of the energy from microtubule depolymerization into processive chromosome motion. The initial focus of our work will be on kinetochore proteins that are not motors. By using previously established approaches that rely on single-molecule fluorescence microscopy, sophisticated laser tweezers, protein-coated microbeads, and electron microscopy, we will extend work already accomplished on both ring-shaped microtubule- binding proteins, like the Dam1 complex (aka DASH) from budding yeasts, and fibrous complexes, like NDC80 (Hec1) from C. elegans, yeasts, and human cells. We are also constructing derivatized planar surfaces, modified by the binding of long, hydrophilic polymers, as platforms for presenting complexes of kinetochore proteins to dynamic microtubule ends, so they can interact in ways that reflect their functions in vivo. Microtubules whose minus ends are firmly tethered to beads will be manipulated with a laser beam to measure the interactions between their labile plus ends and surface-bound kinetochore proteins under controlled, tension-dependent conditions. In cells, however, motor enzymes too have been implicated in proper chromosome attachment to spindle microtubules. Our planar system will be used to investigate functional interactions among couplers and other kinetochore proteins, such as motors and kinases, looking for synergy in the ability of these factors to generate processive movement, to withstand counter forces, and to respond to maladaptive situations. This work should elucidate the mechanisms of chromosome motion, helping us to understand the disease conditions in which accurate chromosome segregation fails, as in the development of aneuploidy. Our work may also help to identify kinetochore proteins that are likely to be valuable targets for the selection of novel drugs that block mitosis.

Public Health Relevance

We are studying the mechanisms by which duplicated chromosomes become attached to the cellular machinery that assures their accurate segregation in preparation for cell division. This cellular process, called "mitosis", is important because its failure leads to daughter cells that have too many or too few chromosomes, a condition associated with advancing cancer and the formation of congenital malformations. Our work will help to identify the molecular functions that are important for accurate chromosome segregation, which may empower future work that corrects or prevents mistakes in this essential cellular activity. We may also help to identify proteins that are ideally suited to serve as targets for the discovery of new drugs that block cell division, one of the strategies that is currently used by some of the effective cancer chemotherapeutics.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Nuclear Dynamics and Transport (NDT)
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Deatherage, James F
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University of Colorado at Boulder
Schools of Arts and Sciences
United States
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Volkov, Vladimir A; Zaytsev, Anatoly V; Grishchuk, Ekaterina L (2014) Preparation of segmented microtubules to study motions driven by the disassembling microtubule ends. J Vis Exp :
Vitre, Benjamin; Gudimchuk, Nikita; Borda, Ranier et al. (2014) Kinetochore-microtubule attachment throughout mitosis potentiated by the elongated stalk of the kinetochore kinesin CENP-E. Mol Biol Cell 25:2272-81
Gudimchuk, Nikita; Vitre, Benjamin; Kim, Yumi et al. (2013) Kinetochore kinesin CENP-E is a processive bi-directional tracker of dynamic microtubule tips. Nat Cell Biol 15:1079-88
Volkov, Vladimir A; Zaytsev, Anatoly V; Gudimchuk, Nikita et al. (2013) Long tethers provide high-force coupling of the Dam1 ring to shortening microtubules. Proc Natl Acad Sci U S A 110:7708-13
McIntosh, J Richard; O'Toole, Eileen; Zhudenkov, Kirill et al. (2013) Conserved and divergent features of kinetochores and spindle microtubule ends from five species. J Cell Biol 200:459-74
McIntosh, J Richard; Molodtsov, Maxim I; Ataullakhanov, Fazly I (2012) Biophysics of mitosis. Q Rev Biophys 45:147-207
Grishchuk, Ekaterina L; Ataullakhanov, Fazly I (2010) In vitro assays to study the tracking of shortening microtubule ends and to measure associated forces. Methods Cell Biol 95:657-76
Grissom, Paula M; Fiedler, Thomas; Grishchuk, Ekaterina L et al. (2009) Kinesin-8 from fission yeast: a heterodimeric, plus-end-directed motor that can couple microtubule depolymerization to cargo movement. Mol Biol Cell 20:963-72
McIntosh, J Richard; Morphew, Mary K; Grissom, Paula M et al. (2009) Lattice Structure of Cytoplasmic Microtubules in a Cultured Mammalian Cell. J Mol Biol :
McIntosh, J Richard; Grishchuk, Ekaterina L; Morphew, Mary K et al. (2008) Fibrils connect microtubule tips with kinetochores: a mechanism to couple tubulin dynamics to chromosome motion. Cell 135:322-33

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