The long term goal is to establish the structural and mechanistic basis for force production by kinesin superfamily members. The proposed studies reflect a longstanding interest in intracellular transport and a commitment to biomedical research.
The specific aims of this research proposal are to establish the kinetic and thermodynamic basis of force generation of the EgS and Kar3 ATPases in direct comparison to Ncd and conventional kinesin. All use ATP to drive unidirectional microtubule based movements. Ncd, Eg5, and Kar3 are involved in spindle dynamics during meiosis and/or mitosis and therefore are required for proper chromosome segregation. In contrast, kinesin is a neuronal motor that promotes movement of membranous organelles. Kinesins motility is distinctive because of its processivity. Ncd, Kar3 and Eg5 are believed not to be processive. Both Ncd and Kar3 promote minus end directed microtubule movements, yet kinesin and Eg5 promote plus end directed movements. Furthermore, Kar3 as a monomer exhibits unidirectional movement; therefore, Kar3 is an interesting motor to study in direct comparison to the climeric kinesins kinesin, Eg5, Ncd. The results with Ncd and kinesin indicate that both motor domains of the dimer are required for movement. Eg5 is also dimeric, yet evidence to date indicates it is not processive. The studies with EgS, in direct comparison to Ncd and kinesin, are intended to define the mechanistic features required specifically for processivity that may be distinct from those features that drive plus end directed movements. In addition, the experiments with mutant kinesin motors will explore aspects of the ATPase crossbridge cycle that are not accessible by studying the wildtype motor. The proposed experiments will evaluate the mechanistic features that spindle motors have in common, and at the same time address specific questions about energy transduction for dimeric motors in comparison to monomeric motors. A comprehensive analysis of these 4 molecular motors will provide information to begin to understand the structural and mechanistic requirements for the diverse movements occurring during the cell cycle and during neuromuscular development and function where genetic alteration can result in birth defects, degenerative diseases, and cancer.

National Institute of Health (NIH)
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Research Scientist Development Award - Research (K02)
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Arthritis and Musculoskeletal and Skin Diseases Special Grants Review Committee (AMS)
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Nuckolls, Glen H
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University of Pittsburgh
Schools of Arts and Sciences
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Chen, Chun Ju; Rayment, Ivan; Gilbert, Susan P (2011) Kinesin Kar3Cik1 ATPase pathway for microtubule cross-linking. J Biol Chem 286:29261-72
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
Cochran, Jared C; Krzysiak, Troy C; Gilbert, Susan P (2006) Pathway of ATP hydrolysis by monomeric kinesin Eg5. Biochemistry 45:12334-44
Sproul, Lisa R; Anderson, Daniel J; Mackey, Andrew T et al. (2005) Cik1 targets the minus-end kinesin depolymerase kar3 to microtubule plus ends. Curr Biol 15:1420-7
Cochran, Jared C; Gatial 3rd, Joseph E; Kapoor, Tarun M et al. (2005) Monastrol inhibition of the mitotic kinesin Eg5. J Biol Chem 280:12658-67

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