TRIM67 regulates growth cone filopodia during netrin-dependent axon guidance

 描述(申请人提供)：轴突指导是中枢神经系统发育的关键一步。越来越多的证据表明，许多患有包括精神分裂症和自闭症在内的多种神经发育障碍的患者的轴突引导受到干扰。引导信号netrin-1和netrin受体DCC通过调节生长锥细胞骨架来调节轴突引导，但其机制尚不完全清楚。我们最近发现E3泛素连接酶TRIM9是DCC的一个新的结合伙伴，是轴突引导和细胞骨架的调节因子。我们也有初步的数据表明，高度相似的蛋白TRIM67与DCC和肌动蛋白调节蛋白Vasp相互作用，是Netrin依赖的轴突分支所必需的。这一提议将检验我们的假设，即TRIM9和TRIM67协同调节netrin-1依赖的轴突引导的下游影响。我们的第一个目标是鉴定和确认TRIM67与DCC和Ena/Vasp肌动蛋白调节蛋白的直接相互作用，以及它们在丝状足端的共定位。我们的第二个目标是确定TRIM67在Netrin依赖的轴突形态发生中的作用。我们的第三个目标将集中在使用新型微流控设备在体外识别TRIM67缺失时发生的轴突引导缺陷，以及使用组织学分析识别发生在体内的缺陷。这些实验将有助于进一步阐明神经元发育的机制。

 DESCRIPTION (provided by applicant): Axon guidance is a critical step in the development of the central nervous system. Mounting evidence suggests that axon guidance is disrupted in many patients suffering from several neurodevelopmental disorders, including schizophrenia and autism. The guidance cue netrin-1 and netrin receptor DCC regulate axon guidance by modulating the growth cone cytoskeleton, but the mechanisms are not fully understood. We recently identified the E3 ubiquitin ligase TRIM9 as a novel binding partner of DCC and a regulator of axon guidance and the cytoskeleton. We also have preliminary data showing that the highly similar protein TRIM67 interacts with DCC and the actin regulatory protein VASP, and is necessary for netrin-dependent axon branching. This proposal will test our hypothesis that TRIM9 and TRIM67 coordinate to regulate the downstream effects of netrin-1 dependent axon guidance. Our first aim will be to identify and confirm TRIM67 direct interactions with DCC and Ena/VASP actin regulatory proteins and their colocalization at filopodia tips. Our second aim is to define the role of TRIM67 in netrin-dependent axon morphogenesis. Our third aim will focus on identifying axon guidance defects that occur upon deletion of TRIM67 in vitro, using novel microfluidic devices, as well as defects that occur in vivo using histological analysis. These experiments will help to further elucidate mechanisms of neuronal development.