Molecular mechanism of Kinesin-2 motility

描述（由申请人提供）：分子马达的驱动蛋白-2家族在称为鞭毛内转运的过程中沿沿着和鞭毛转运货物。鞭毛内运输的中断导致细胞水平的纤毛和鞭毛截短，并且在生物体水平导致身体计划发育和器官衰竭的缺陷。有一个共同的努力正在进行中，以确定鞭毛内运输的分子机制，但在分子尺度上表征这一过程是困难的，几乎完全缺乏了解的分子机制的驱动蛋白-2电机。为了回答诸如运动活动如何调节以及运输需要多少马达等细胞问题，必须首先在单分子水平上表征驱动蛋白-2马达，并定义其机制基础的关键生化转变。驱动蛋白2马达的一个独特而令人困惑的特征是，它们不是像大多数驱动蛋白那样包含两个相同的马达结构域，而是由两个不同的马达结构域组成。在PI实验室进行的结构域交换实验现在已经表明，这两个头部以不同的速度移动，导致假设它们的活动被调整以优化完整异二聚体的运输特性。 拟开展的工作的目标是通过表征KIF 3A/B（小鼠驱动蛋白-2的直系同源物）来确定驱动蛋白-2功能的分子机制。单分子荧光和光镊实验将测量这些马达的性能特征（速度，力的产生和微管亲和力），并将揭示这些马达的内部工作原理，以揭示运动和调节的结构基础。具体目的如下：1）测试两个头是否以不同的速率沿沿着行进。2)确定动力学差异是否源于运动域的结构差异或运动域之间的协调。3)测量马达在每次遇到微管时所采取的连续步骤的数量，以及这种持续合成能力对外部负载的依赖性。4)确定驱动蛋白-2动力学循环中控制运动速度和持续合成能力的生化转变。实验数据将被纳入驱动蛋白-2运动的计算模型，以测试有关这些电机的细胞行为的假设。 了解驱动蛋白-2运动的分子机制对于揭示基于转运的疾病如多囊肾病、精子运动缺陷和视网膜变性的分子基础是重要的。此外，深入了解驱动蛋白马达的机械化学将有助于开发针对有丝分裂驱动蛋白的抗肿瘤疗法。

DESCRIPTION (provided by applicant): The Kinesin-2 family of molecular motors transport cargo along cilia and flagella in a process termed intraflagellar transport. Disruption of intraflagellar transport results in truncated cilia and flagella at the cellular level, and at the organismal level leads to defects in body plan development and organ failure. There is a concerted effort underway to define the molecular machinery underlying intraflagellar transport, but characterizing this process at the molecular scale is made difficult by the virtually complete lack of understanding of the molecular mechanism of the Kinesin-2 motor. To answer cellular questions such as how the motor activity is regulated and how many motors are required for transport, it is essential to first characterize Kinesin-2 motors at the single-molecule level, and define the key biochemical transitions that underlie their mechanism. A unique and puzzling feature of Kinesin-2 motors is that instead of containing two identical motor domains like most kinesins, they are made up of two different motor domains. Domain swapping experiments carried out in the PI's lab have now shown that these two heads move at different speeds, leading to the hypothesis that their activities are tuned to optimize transport characteristics of the intact heterodimer. The goal of the proposed work is to define the molecular mechanism of Kinesin-2 function by characterizing KIF3A/B, the mouse Kinesin-2 ortholog. Single-molecule fluorescence and optical tweezer experiments will measure the performance characteristics of these motors (speed, force production and microtubule affinity) and will uncover the inner workings of these motors to reveal the structural basis of movement and regulation. The specific aims are as follows: 1) Test whether the two heads step along microtubules at different rates. 2) Determine whether kinetic differences arise from structural differences in the motor domains or from coordination between the motor domains. 3) Measure the number of sequential steps motors take during each encounter with a microtubule and the dependence of this processivity on external load. 4) Identify the biochemical transitions in the Kinesin-2 kinetic cycle that control motor speed and processivity. Experimental data will be incorporated into computational models of Kinesin-2 motility to test hypotheses regarding the cellular behavior of these motors. Understanding the molecular mechanism of Kinesin-2 motility is important for uncovering the molecular basis of transport-based diseases such as polycystic kidney disease, defects in sperm motility, and retinal degeneration. Furthermore, insights into the mechanochemistry of kinesin motors will aid in developing anti- tumor therapies targeting mitotic kinesins.