A major challenge in biomedical research is to define the mechanisms for mitotic spindle assembly and how a complex array of motors and microtubule (MT) interacting proteins correctly orchestrate chromosome segregation. Defects in mitosis result in birth defects and cancer, and therefore, a full molecular understanding of mitotic mechanisms is critical for human development and health. Mitotic kinesins play essential roles in all facets of spindle function- effecting chromosome movement and segregation, cell cleavage, and regulating microtubule polymerization and depolymerization. While kinesins share common structural motifs, key amino acid changes confer unique mechanochemical properties to each kinesin which specifies its cellular function. Therefore, if we elucidate the enzymatic properties of an individual mitotic kinesin in vitro, we will gain insight into its specific role for spindle function in the complex environment of the cell where there are other molecular motors, proteins, and regulatory factors. The research proposed evaluates three representative kinesins to address questions about the mechanistic requirements for processive movement of chromosomes on the microtubule lattice, MT-MT crosslinking function for MT sliding and spindle stability, and MT shortening and MT elongation for spindle assembly and dynamics. We will use presteady-state kinetic methodologies in combination with equilibrium approaches and fluorescence microscopy to address the following three specific aims: 1) Define the Kar3Cik1 mechanochemistry for its crosslinking function of anti-parallel MTs at anaphase. 2) Define the Kar3Vik1 mechanochemistry for its accumulation at MT minus-ends to crosslink parallel MTs at the spindle poles. 3) Define the mechanistic basis of CENP-E processive stepping.
CENP-E, Eg5/KSP, and Kinesin-14s are essential for cell division and therefore human health and development. Their selective inhibition may lead to more effective anti-mitotic therapeutics for treatment of diseases such as cancer, symptomatic coronary artery disease, and proliferative diabetic retinopathy. The proposed studies should lead to new strategies for high throughput screens that select compounds to enhance cancer cell death rather than aneuploidy after chemotherapy. Presently, there are a number of specific chemotherapeutics targeted to Eg5/KSP and CENP-E in Phase I/II Clinical trials.
|Rayment, Ivan (2014) Structural insights into the assembly of a monomeric class V myosin. Proc Natl Acad Sci U S A 111:4351-2|
|Cope, Julia; Rank, Katherine C; Gilbert, Susan P et al. (2013) Kar3Vik1 uses a minus-end directed powerstroke for movement along microtubules. PLoS One 8:e53792|
|Chen, Chun Ju; Rayment, Ivan; Gilbert, Susan P (2011) Kinesin Kar3Cik1 ATPase pathway for microtubule cross-linking. J Biol Chem 286:29261-72|
|Sardar, Harjinder S; Luczak, Vincent G; Lopez, Maria M et al. (2010) Mitotic kinesin CENP-E promotes microtubule plus-end elongation. Curr Biol 20:1648-53|
|Krzysiak, Troy C; Grabe, Michael; Gilbert, Susan P (2008) Getting in sync with dimeric Eg5. Initiation and regulation of the processive run. J Biol Chem 283:2078-87|
|Valentine, Megan T; Gilbert, Susan P (2007) To step or not to step? How biochemistry and mechanics influence processivity in Kinesin and Eg5. Curr Opin Cell Biol 19:75-81|
|Allingham, John S; Sproul, Lisa R; Rayment, Ivan et al. (2007) Vik1 modulates microtubule-Kar3 interactions through a motor domain that lacks an active site. Cell 128:1161-72|
|Krzysiak, Troy C; Wendt, Thomas; Sproul, Lisa R et al. (2006) A structural model for monastrol inhibition of dimeric kinesin Eg5. EMBO J 25:2263-73|
|Krzysiak, Troy C; Gilbert, Susan P (2006) Dimeric Eg5 maintains processivity through alternating-site catalysis with rate-limiting ATP hydrolysis. J Biol Chem 281:39444-54|
|Hertzer, Kathleen M; Ems-McClung, Stephanie C; Kline-Smith, Susan L et al. (2006) Full-length dimeric MCAK is a more efficient microtubule depolymerase than minimal domain monomeric MCAK. Mol Biol Cell 17:700-10|
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