Molecular Mechanism of Kinesin-2 Motility

描述(申请人提供)：Kinesin-2马达在纤毛和鞭毛中进行顺行运输，以及在细胞中进行其他双向运输过程。虽然机械力化学中运动的许多方面已经被理解，但作为多电机和双向货物运输基础的特定的电机活动和调节还不是很清楚。Kinesin-2转运不足会导致发育异常、光感受器降解和多囊肾病。该项目的目标是了解Kinesin-2马达沿着微管行走的机制，以及Kinesin-2马达属性在多大程度上针对其特定的运输任务进行调整。与经典的Kinesin-1马达家族的显著不同是，连接核心马达头部和螺旋线圈结构域的Kinesin-2的颈部连接结构域有17个氨基酸，而Kinesin-1只有14个氨基酸。由于颈部连接件用作将每个头部连接到其共享线圈线圈的机械元件，因此延伸颈部连接件预计将减弱头部区域之间的机械力化学耦合。与此一致，我们发现延长kinesin-1颈部连接子会降低其加工性，而缩短kinesin-2颈部连接子会增强加工性。此外，我们发现Kinesin-2的速度和运行长度与Kinesin-1的力依赖关系不同，这表明电机可能被优化为双向货物运输而不是长距离的单向运输。利用单分子和多运动实验，结合化学机械循环中运动蛋白的计算模型，我们将揭示运动蛋白-1和运动蛋白-2马达之间机制差异的结构基础，目的是了解体内的双向运输。这项工作包括结构研究，以确定颈部连接子和颈部线圈结构域在沿微管的前进运动中所起的作用。我们还将研究kinesin-2化学机械循环中的具体步骤，以确定运动生物化学如何由两个头域之间的分子内张力以及光钳施加的外部负载控制。这些对Kinesin-2运动机制的研究将为理解Kinesin-2在细胞中的运输提供一个框架。通过以kinesin-1为基准，这些测量将建立kinesin机械化学的普遍主题，这将有助于更好地了解神经退行性疾病的分子基础，并有助于开发针对有丝分裂kinesins的抗肿瘤治疗。

DESCRIPTION (provided by applicant): Kinesin-2 motors carry out anterograde transport in cilia and flagella, as well as other bidirectional transport processes in cells. Although many aspects of kinesin mechanochemistry are understood, the specific motor activities and regulation that underlie multi-motor and bidirectional cargo transport are not well understood. Deficiencies in Kinesin-2 transport lead to abnormal development, photoreceptor degradation and polycystic kidney disease. The goals of this project are to understand the mechanism by which Kinesin-2 motors walk along microtubules, and the degree to which Kinesin-2 motor properties are tuned for its specific transport tasks. A notable difference from the canonical Kinesin-1 motor family is that neck linker domain of Kinesin-2, which connects the core motor head to the coiled-coil domain, is 17 amino acids compared to only 14 in Kinesin-1. Because the neck linker serves as a mechanical element connecting each head to their shared coil-coil, it is expected that extending the neck linker will diminish mechanochemical coupling between the head domains. Consistent with this, we found that extending the Kinesin-1 neck linker diminishes its processivity and shortening the Kinesin-2 neck linker enhances processivity. In addition, we found that the force dependence of Kinesin-2 velocity and run length differ from Kinesin-1, suggesting the motor may be optimally tuned for bidirectional cargo transport rather than long distance unidirectional transport. Using single-molecule and multi-motor experiments in conjunction with computational modeling of the kinesin chemomechanical cycle, we will uncover the structural basis of mechanistic differences between Kinesin-1 and Kinesin-2 motors, with the goal of understanding bidirectional transport in vivo. This work involves structural studies to determine the role played by the neck linker and neck coil domains in processive movement along microtubules. We will also investigate specific steps in the Kinesin-2 chemomechanical cycle to determine how the motor biochemistry is controlled by intramolecular tension between the two head domains, as well as by external loads applied by optical tweezers. These investigations into the mechanism of Kinesin-2 motility will provide a framework in which to understand Kinesin-2 transport in cells. By benchmarking against Kinesin-1, these measurements will establish universal themes underlying kinesin mechanochemistry that will help to better understand the molecular basis of neurodegenerative diseases and aid the effort to develop anti-tumor therapies targeting mitotic kinesins.