Mechanistic Analysis of Microtubule Based Motors

总体目标是获得关键的见解的基本原理驱动蛋白运动结构， 功能，并将这种理解外推到细胞的内部工作。驱动蛋白超家族成员 共享一个共同的催化结构域，但参与广泛的细胞功能， 运输、有丝分裂和减数分裂、微管动力学的调节以重塑细胞骨架，以及 细胞极性的产生。现在认识到序列差异改变机械化学， 微管相互作用，以及对力的反应，其中每一个对于特定的生理过程都是至关重要的。 功能本提案的目标是建立驱动蛋白共有的机制和结构特征， 14 Kar 3Cik 1，Kar 3Vik 1和Ned，同时揭示了导致功能性 的特异性驱动蛋白-14亚家族的成员是已知促进微管减- 端部定向力的产生和使用ATP促进动力冲程机制。相反，成员 驱动蛋白-1，2，5和7亚家族的分子产生微管加末端导向力，并且这些分子 发动机是连续的。常规驱动蛋白-1、驱动蛋白-5 Eg 5和驱动蛋白-7 CENP-E产生二聚体 马达来自相同的基因产物，但驱动蛋白-2的KIF 3AB和KIF 3AC的功能性催化二聚体 由两种不同的基因产物产生。因此，异二聚体催化的选择性优势是什么？ 对于体内功能的酶，头-头通信是如何建立的，以调节与 微管晶格和/或微管末端，以及什么机制调节持续合成能力和 对武力的回应？这项研究提出了评估异二聚体Kar 3Cik 1和Kar 3Vik 1， 同源二聚体Ned和异源二聚体驱动蛋白-2 KIFAB和KIFAC与其他进行性 同源二聚体驱动蛋白。实验方法包括稳态前动力学方法、单稳态动力学方法、单稳态动力学方法和单稳态动力学方法。 分子和整体荧光显微镜，光学捕获，以确定力依赖的运动 性质，X射线晶体学，低温电子显微镜和断层扫描，以及计算建模。 这种全面的分析将提供新的见解，以了解机械化学的基础， 细胞组织和生理功能所需的结构-功能关系。 相关性（参见说明）： 总的目标是了解驱动蛋白马达的机械化学，这是它们的能力的基础， 促进细胞内运输，细胞极性的产生，以及微管细胞骨架的重塑， 细胞分裂、细胞分化和人类发育过程中的形态发生。驱动蛋白的缺陷 与多种病理学有关，包括癌症、纤毛病、神经病和出生缺陷。

The overarching goal is to gain critical insights into the fundamentals of kinesin 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. It is now recognized that sequence differences modify the mechanochemistry, microtubule interactions, and the response to force, each of which is critical for the specific physiological function. The goal of this proposal is to establish the mechanistic and structural features shared by kinesin- 14 Kar3Cik1, Kar3Vik1, and Ned 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 to use an ATP-promoted powerstroke mechanism. In contrast, members of kinesin-1, 2, 5, and 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 dimeric motors from the same gene product, yet the functional catalytic dimer for kinesin-2 KIF3AB and KIF3AC arises from two different gene products. Therefore, what is the selective advantage of heterodimeric catalytic enzymes for in vivo function, how is head-head communication established to modulate interactions with the microtubule lattice and/or microtubule end, and what mechanisms regulate the interplay of processivity and response to force? The research proposed evaluates heterodimeric Kar3Cik1 and Kar3Vik1 in comparison to homodimeric Ned, and heterodimeric Kinesin-2 KIFAB and KIFAC in comparison to other processive homodimeric kinesins. Experimental approaches include presteady-state kinetics methodologies, single molecule and ensemble fluorescence microscopy, optical trapping to determine the force-dependent motility properties, X-ray crystallography, cryo-electron microscopy and tomography, and computational modeling. This comprehensive analysis will provide new insights to understand the mechanochemistry that underlies structure-function relationships required for cellular organization and physiological function. RELEVANCE (See instructions): The overall goal is to understand the mechanochemistry of kinesin motors that underlies their ability to promote intracellular transport, generation of cell polarity, and remodeling of the microtubule cytoskeleton for cell division, cell differentiation, and morphogenesis during human development. Defects in kinesins have been linked to diverse pathologies including cancer, ciliopathies, neuropathies, and birth defects.