Mechanistic Analysis of Microtubule Based Motors

描述（由申请人提供）：总体目标是获得对运动结构和功能的基本原理的重要见解，并将这种理解外推到细胞的内部运作。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，包括细胞内运输，有丝分裂和减数分裂，微管动力学的调节，用于细胞骨架的重塑，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化学和微管相互作用。本研究的目的是建立驱动蛋白14 Kar 3Cik 1、Kar 3Vik 1和Ncd共同的机制和结构特征，同时揭示导致功能特异性的独特特征。驱动蛋白-14亚家族的成员是已知促进微管负末端定向力产生的唯一驱动蛋白，并且这些马达不是进行性的。相比之下，Kinesin-1，2，5，7亚家族的成员产生微管加末端导向力，并且这些分子马达是进行性的。传统的驱动蛋白-1、驱动蛋白-5 Eg 5和驱动蛋白-7 CENP-E从相同的基因产物产生马达，而驱动蛋白-2的功能性催化二聚体来自两种不同的基因产物。因此，异源二聚体催化酶在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？该研究提出了与同型二聚体Ncd相比的异源二聚体Kar 3Cik 1和Kar 3Vik 1，以及与其他进行性同型二聚体驱动蛋白（包括常规驱动蛋白-1，Eg 5和CENP-E）相比的异源二聚体驱动蛋白-2 KIFAB和KIFAC。实验方法包括预稳态动力学方法、微管-马达复合体的荧光显微镜、X射线晶体学、以及低温电子显微镜和断层摄影术。这种对驱动蛋白-14和2成员的全面分析将为理解细胞组织和功能所需的结构-功能关系的机械化学提供新的见解。

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.