超长的轴突结构为长距离的神经连接提供了基础，然而长的轴突对分子马达将膜及相关蛋白运输至轴突末端提出了更高的要求。轴突物质运输的缺陷会导致神经疾病如神经退行性疾病等。驱动蛋白和动力蛋白是轴突运输中的主要分子马达，其调节机制还知之甚少。通过遗传筛选我们发现小G蛋白ARL-8对驱动蛋白KIF1a/UNC-104的激活是必要的。ARL-8突变导致突触囊泡和活性区蛋白错误的位于近端轴突，而过表达ARL-8使得突触囊泡被运输到更远的位置。ARL-8自主性的发挥作用并且定位于突触囊泡上，而突触囊泡正是UNC-104驱动蛋白的运输货物。ARL-8在序列和功能上极其保守的。我们通过arl-8突变体抑制基因筛选得到了一系列的UNC-104突变体，它们导致驱动蛋白的活性高于野生型。我们将从遗传、细胞、生化和结构生物学等方面研究这些突变对驱动蛋白的影响，以理解驱动蛋白的调节机制，也为相关疾病研究提供线索。

The extraordinary length of axons affords long-range neuronal connectivity, which is essential for the evolution of large animals. However, long axons create high demands for molecular motors to shuttle membrane and protein material between axonal terminal and neuronal cell body. Defects in axonal transport give rise to congenital neurological symptoms and contribute to many neurodegenerative diseases. While the kinesins and dynein family of proteins have been identified as key molecular motors responsible for the axonal transport, the regulation of these motors are poorly understood. We have established in vivo and in vitro experimental systems to study the regulation of kinesin. Using forward genetic screens in C. elegans, we discovered a small Gprotein ARL-8 is critical to stimulate the activity of the kinesin KIF1a/UNC-104. Loss of ARL-8 activity causes synaptic vesicles and active zone proteins to mislocalize in proximal axon segments. Overexpression of ARL-8 causes synaptic vesicle to travel too far. ARL-8 is extremely conserved both at the sequence and functional level. ARL-8 functions cell autonomously in neurons and localize on the synaptic vesicles: cargoes for the UNC-104 kinesin..In a genetic suppressor screen in the arl-8 mutant background, we identified a series of mutations in UNC-104. Preliminary analyses show that all of these mutations are “gain-of-function” mutations that render the kinesin “higher than normal” activity. In this grant, I propose to investigate the biological consequences of these mutated kinesin both in vivo and in vitro to understand how kinesin molecules are regulated. Specifically, we will ask the following questions:.1. What are the in vivo trafficking phenotypes caused by these gain-of-function mutations in unc-104. We will measure the speed, pause, run length of kinesin-mediated movements as well as the synaptic parameters..2. How do these mutations generate a “gain-of-function”? We will collaborate with Dr. Wei Feng’s lab at Institute of Biophysics (where I am a part time faculty) to understand the biochemical and biophysical properties of these mutant motors..3. How does ARL-8 regulate UNC-104? We will extend our biochemical analyses between ARL-8 and UNC-104 to fully understand the mechanisms of ARL-8’s regulation of UNC-104..

分子马达蛋白通过水解ATP提供能量沿着微管运输“货物”至细胞的不同部位。体外研究表明许多分子马达存在分子内自抑制，然而其生理意义未知。我们发现了KIF1A/UNC-104的四个位于马达结构域和杆状（stalk）区域的突变体可以特异地破坏自抑制，体外研究表明这些突变体可以增强和微管、囊泡的结合。在体研究表明，它们可以导致UNC-104的过度活化，引起突触密度下降，突触变小和树突异位突触囊泡出现。进一步我们发现突触囊泡结合的小G蛋白ARL-8可以激活UNC-104，这是通过解锁其自抑制实现的。总之，马达蛋白自抑制机制可以调控运输“货物”的分布，这对突触发生非常重要。