The broad goal of this project is to understand how dynamic microtubules are controlled during cell division to build the mitotic spindle and to segregate chromosomes with high fidelity. This project is designed to investigate the mechanism by which mitotic microtubule motor proteins of the kinesin family regulate microtubule dynamics in order to facilitate mitotic spindle assembly, chromosome segregation and long-term stability of chromosome number. Our approach is interdisciplinary, ranging from single molecule biophysical analyses using total internal reflection microscopy (TIRF) and purified components to cellular approaches using high resolution quantitative live cell imaging, mutant constructs and siRNA depletions. We will use TIRF microscopy of live microtubules at physiological temperature and buffer conditions to evaluate the effect of mitotic kinesins on microtubule assembly and disassembly. Armed with an understanding of the activity the motor provides to microtubules in isolation we will transfer our studies to live mitotic cells. Using hig resolution imaging, selective depletions of kinesins and mutant analogs we will evaluate how kinesin motor activity is used within the mitotic spindle. This is essential basic knowledge which will guide the development of drugs targeted to mitotic kinesins for use in cancer treatment. Finally, we have preliminary evidence that small alterations in microtubule dynamics that do not have a profound impact on cell division in the short term have significant effects on long-term chromosome instability. Furthermore, known tumor suppressor genes which have not previously been linked to microtubules may exhibit unexpected direct or indirect effects on microtubule dynamics. We have a simple visual screen that will provide us with a panel of genes implicated in microtubule dynamics alterations and, by extension, chromosome instability. This will assist in molecular characterization of cancers and targeted drug development. Using these approaches we will learn how motile kinesins target to and regulate polymer assembly and disassembly at microtubule ends and what effect alterations in microtubule dynamics has on short-term spindle assembly and chromosome segregation. This project is designed to evaluate the effect that small changes in microtubule dynamics in the mitotic spindle has on chromosome instability and, in this way, understand the mechanistic forces underlying tumor progression.

Public Health Relevance

During cell division chromosomes must be segregated to each daughter cell with perfect fidelity to avoid the alterations in chromosome numbers characteristic of cancer cells. This process is critically dependent on dynamic polymers within the cell called "microtubules" and cells have evolved numerous motor molecules that regulate microtubule assembly and disassembly. By investigating the mechanism of action of these regulators we will uncover new, tissue-specific targets for cancer therapies and increase our understanding of the molecular events that drive cell division in normal and cancer cells.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM069429-09
Application #
8368580
Study Section
Cellular Signaling and Regulatory Systems Study Section (CSRS)
Program Officer
Deatherage, James F
Project Start
2004-08-01
Project End
2016-07-31
Budget Start
2012-09-01
Budget End
2013-07-31
Support Year
9
Fiscal Year
2012
Total Cost
$377,640
Indirect Cost
$119,236
Name
University of Washington
Department
Physiology
Type
Schools of Medicine
DUNS #
605799469
City
Seattle
State
WA
Country
United States
Zip Code
98195
Drum, Benjamin M L; Yuan, Can; Li, Lei et al. (2016) Oxidative stress decreases microtubule growth and stability in ventricular myocytes. J Mol Cell Cardiol 93:32-43
Wordeman, Linda; Decarreau, Justin (2016) Revisiting Actin's role in early centrosome separation. Cell Cycle 15:162-3
Luo, Ruibai; Chen, Pei-Wen; Wagenbach, Michael et al. (2016) Direct Functional Interaction of the Kinesin-13 family membrane Kinesin Like Protein 2A (Kif2A) and Arf GAP with GTP-Binding Protein-Like, Ankyrin Repeats and PH domains1 (AGAP1). J Biol Chem :
Wordeman, Linda; Decarreau, Justin; Vicente, Juan Jesus et al. (2016) Divergent microtubule assembly rates after short- versus long-term loss of end-modulating kinesins. Mol Biol Cell 27:1300-9
Cherry, Allison E; Haas, Brian R; Naydenov, Alipi V et al. (2016) ST-11: A New Brain-Penetrant Microtubule-Destabilizing Agent with Therapeutic Potential for Glioblastoma Multiforme. Mol Cancer Ther 15:2018-29
Vicente, Juan Jesus; Wordeman, Linda (2015) Mitosis, microtubule dynamics and the evolution of kinesins. Exp Cell Res 334:61-9
Hehnly, Heidi; Canton, David; Bucko, Paula et al. (2015) A mitotic kinase scaffold depleted in testicular seminomas impacts spindle orientation in germ line stem cells. Elife 4:e09384
Burns, Kyle M; Sarpe, Vladimir; Wagenbach, Mike et al. (2015) HX-MS2 for high performance conformational analysis of complex protein states. Protein Sci 24:1313-24
Rey, Martial; Sarpe, Vladimir; Burns, Kyle M et al. (2014) Mass spec studio for integrative structural biology. Structure 22:1538-48
Decarreau, Justin; Driver, Jonathan; Asbury, Charles et al. (2014) Rapid measurement of mitotic spindle orientation in cultured mammalian cells. Methods Mol Biol 1136:31-40

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