Molecular mechanism of Kinesin-2 motility

描述(由申请人提供)：Kinesin-2分子马达家族沿着纤毛和鞭毛运输货物，这一过程称为鞭毛内运输。鞭毛内运输的中断会导致细胞水平的纤毛和鞭毛被截断，在生物水平上会导致身体计划发育的缺陷和器官衰竭。人们正在共同努力定义鞭毛内转运的分子机制，但由于几乎完全缺乏对Kinesin-2马达的分子机制的了解，在分子水平上表征这一过程是困难的。为了回答细胞问题，如马达活动是如何调节的，运输需要多少马达，必须首先在单分子水平上表征Kinesin-2马达，并定义其机制下的关键生化转变。Kinesin-2马达的一个独特而令人费解的特征是，它们不像大多数Kinesin那样包含两个相同的运动域，而是由两个不同的运动域组成。在PI的实验室中进行的结构域交换实验现在已经表明，这两个头以不同的速度移动，这导致了他们的活动被调整以优化完整的异二聚体的运输特性的假设。 这项工作的目的是通过鉴定KIF3A/B，小鼠Kinesin-2的同源基因，来确定Kinesin-2功能的分子机制。单分子荧光和光钳实验将测量这些马达的性能特征(速度、作用力和微管亲和力)，并将揭示这些马达的内部工作方式，以揭示运动和调节的结构基础。具体目的如下：1)测试两个头部是否以不同的速度沿着微管行走。2)确定运动学差异是由运动域的结构差异还是运动域之间的协调引起的。3)测量电机在每次与微管相遇时采取的连续步数，以及这种过程对外部负载的依赖程度。4)确定Kinesin-2动力学周期中控制马达速度和加工能力的生化转变。实验数据将被合并到Kinesin-2运动性的计算模型中，以测试关于这些马达的细胞行为的假设。 了解Kinesin-2运动的分子机制对于揭示多囊肾病、精子运动障碍和视网膜退化等基于转运的疾病的分子基础非常重要。此外，对动蛋白马达的机械性化学的深入了解将有助于开发针对有丝分裂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.