TRIM67 regulates growth cone filopodia during netrin-dependent axon guidance

 描述（由申请人提供）：轴突引导是中枢神经系统发育的关键步骤。越来越多的证据表明，在许多患有几种神经发育障碍的患者中，包括精神分裂症和自闭症，轴突导向被破坏。导向因子netrin-1和netrin受体DCC通过调节生长锥细胞骨架来调节轴突导向，但其机制尚不完全清楚。我们最近确定了E3泛素连接酶TRIM 9作为一种新的结合伙伴的DCC和调节轴突的指导和细胞骨架。我们也有初步的数据表明，高度相似的蛋白TRIM 67与DCC和肌动蛋白调节蛋白VASP相互作用，是必要的netrin依赖性轴突分支。该提议将检验我们的假设，即TRIM 9和TRIM 67协调调节netrin-1依赖性轴突导向的下游效应。我们的第一个目标将是确定和确认TRIM 67与DCC和Ena/VASP肌动蛋白调节蛋白的直接相互作用及其在丝状伪足尖端的共定位。我们的第二个目标是确定TRIM 67在netrin依赖性轴突形态发生中的作用。我们的第三个目标将集中在识别轴突导向缺陷时发生的TRIM 67在体外删除，使用新的微流体设备，以及缺陷发生在体内使用组织学分析。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.