Mechanistic Analysis of Microtubule Based Motors

描述(由申请人提供)：首要目标是获得对马达结构和功能的基本原理的批判性见解，并将这一理解外推到细胞的内部工作。Kinesin超家族成员共享一个共同的催化域，但参与了广泛的细胞功能，包括细胞内运输、有丝分裂和减数分裂，调节细胞骨架重构的微管动力学，以及细胞极性的产生。序列差异改变了对特定功能至关重要的机械力化学和微管相互作用。该方案的目的是建立Kinesin-14Kar3Cik1、Kar3Vik1和NCD所共有的机制和结构特征，同时揭示导致功能特异性的独特特征。Kinesin-14亚家族成员是已知的唯一促进微管负端定向作用力产生的Kinesin，这些马达不是过程性的。相反，Kinesin-1、2、5、7亚家族的成员产生微管+末端定向力，这些分子马达是进行性的。传统的Kinesin-1、Kinesin-5 EG5和Kinesin-7 CENP-E产生的马达来自同一个基因产物，而Kinesin-2的功能性催化二聚体则来自两个不同的基因产物。因此，异源二聚体催化酶对体内功能的选择性优势是什么？头-头通讯是如何建立的，以调节与微管晶格和/或微管末端的相互作用？建议的研究将异二聚体Kar3Cik1和Kar3Vik1与同源二聚体NCD进行比较，并将异二聚体kinesin-2 KIFAB和KIFAC与包括常规kinesin-1、EG5和CENP-E在内的其他过程中的同源二聚体kinesin进行比较。实验方法包括稳态前动力学方法、微管-运动复合体的荧光显微镜、X射线结晶学、冷冻电子显微镜和层析成像。这种对Kinesin-14和Kinesin-2成员的全面分析将为理解细胞组织和功能所需的结构-功能关系所依据的机械力化学提供新的见解。

DESCRIPTION (provided by applicant): The overarching goal is to gain critical insights into the fundamentals of motor structure and function and to extrapolate this understanding to the inner workings of the cell. Kinesin superfamily members share a common catalytic domain yet participate in a wide range of cellular functions including intracellular transport, mitosis and meiosis, regulation of microtubule dynamics for remodeling of the cytoskeleton, and generation of cell polarity. Sequence differences modify the mechanochemistry and microtubule interactions that are critical for the specific function. The goal of this proposal is to establishthe mechanistic and structural features shared by Kinesin-14 Kar3Cik1, Kar3Vik1, and Ncd and at the same time to reveal unique features that result in functional specificity. Members of the Kinesin-14 subfamily are the only kinesins known to promote microtubule minus-end-directed force generation, and these motors are not processive. In contrast, members of Kinesin-1, 2, 5, 7 subfamilies generate microtubule plus-end-directed force, and these molecular motors are processive. Conventional Kinesin-1, Kinesin-5 Eg5, and Kinesin-7 CENP-E generate motors from the same gene product, yet the functional catalytic dimer for Kinesin-2 arises from two different gene products. Therefore, what is the selective advantage of heterodimeric catalytic enzymes for in vivo function and how is head-head communication established to modulate interactions with the microtubule lattice and/or microtubule end? The research proposed evaluates heterodimeric Kar3Cik1 and Kar3Vik1 in comparison to homodimeric Ncd, and heterodimeric Kinesin-2 KIFAB and KIFAC in comparison to other processive homodimeric kinesins including conventional Kinesin-1, Eg5, and CENP-E. Experimental approaches include pre-steady state kinetic methodologies, fluorescence microscopy of microtubule-motor complexes, X-ray crystallography, and cryo- electron microscopy and tomography. This comprehensive analysis of Kinesin-14 and Kinesin-2 members will provide new insights to understand the mechanochemistry that underlies structure-function relationships required for cellular organization and function.