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，这一过程被称为鞭毛内运输。鞭毛内运输的中断在细胞水平上导致纤毛和鞭毛的截短，在组织水平上导致身体发育缺陷和器官衰竭。目前，人们正在努力确定筋膜内运输的分子机制，但由于对Kinesin-2马达的分子机制几乎完全缺乏了解，在分子尺度上描述这一过程变得很困难。为了回答诸如运动活动是如何调节的以及运输需要多少运动等细胞问题，首先必须在单分子水平上表征Kinesin-2运动，并定义其机制基础的关键生化转变。运动蛋白-2马达的一个独特而令人费解的特点是，它不像大多数运动蛋白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实验室进行的区域交换实验表明，这两个头部以不同的速度移动，这导致了一种假设，即它们的活动被调整以优化完整异二聚体的运输特性。

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.