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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM033787-27
Application #
8101338
Study Section
Nuclear Dynamics and Transport (NDT)
Program Officer
Gindhart, Joseph G
Project Start
1984-12-01
Project End
2013-06-30
Budget Start
2011-07-01
Budget End
2012-06-30
Support Year
27
Fiscal Year
2011
Total Cost
$453,871
Indirect Cost
Name
University of Colorado at Boulder
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
007431505
City
Boulder
State
CO
Country
United States
Zip Code
80309
Giddings Jr, Thomas H; Morphew, Mary K; McIntosh, J Richard (2017) Preparing Fission Yeast for Electron Microscopy. Cold Spring Harb Protoc 2017:pdb.prot091314
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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) Cryoelectron Microscopy of Fission Yeast. Cold Spring Harb Protoc 2017:pdb.prot091330
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:pdb.prot091322
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
McIntosh, J Richard; Morphew, Mary K; Giddings Jr, Thomas H (2017) Electron Microscopy of Fission Yeast. Cold Spring Harb Protoc 2017:pdb.top079822
McIntosh, J Richard; Hays, Thomas (2016) A Brief History of Research on Mitotic Mechanisms. Biology (Basel) 5:
McIntosh, J Richard (2016) Mitosis. Cold Spring Harb Perspect Biol 8:
Gergely, Zachary R; Crapo, Ammon; Hough, Loren E et al. (2016) Kinesin-8 effects on mitotic microtubule dynamics contribute to spindle function in fission yeast. Mol Biol Cell 27:3490-3514

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