Molecular analysis of the chaperone Hsc70-Trio protein complex in the neuronal growth cone

在发育过程中，神经元的寻路受到指导信号的控制，例如分泌蛋白Netrin-1。Netrin-1与结直肠癌(DCC)中缺失的受体结合，表达在轴突末端的神经元生长锥上，启动分子信号，调节和改变细胞骨架动力学，导致轴突生长和引导。我们发现，鸟嘌呤核苷酸交换因子三联体是生长锥引导中netrin-1/DCC信号的关键组成部分。Trio是一个大的(330 KDa)的细胞质蛋白，含有三个催化区，包括一个丝氨酸/苏氨酸激酶区和两个分别作为rac1和RhoA激活剂的GEFD1和GEFD2。Trio包含其他基序，包括Sec14脂结合结构域、8个血影蛋白重复序列、2个Src同源3结构域和一个免疫球蛋白样结构域。这些不同结构域的存在表明，Trio作为多个上游输入的整合者和多个下游通路的激活器发挥作用。然而，由于缺乏对蛋白质结构的了解，对Trio如何整合这些信号的了解受到限制。为了研究Netrin-1/DCC下游调控Trio定位和激活的机制，我们采用蛋白质组学的方法，确定分子伴侣Hsc70是发育中皮质中一个新的Trio调节因子。此外，我们还鉴定了四个三聚体磷酸化丝氨酸残基，其确切功能仍有待阐明。 总体而言，我们最近的工作强调了Trio在神经发育中的重要作用。然而，Trio调节轴突对引导线索的反应的分子机制仍然不清楚。因此，本研究计划的目标是研究Trio蛋白复合体在神经元生长锥的调节、定位和分子结构，具体有三个目的： 目的1：研究丝氨酸磷酸化在Trio功能和调控中的作用 目的2：用BioID方法鉴定轴突生长中的整体Trio互动组 目的3.利用高分辨率分子低温电子显微镜(Cryo-EM)结合荧光显微镜(Relative Light Electronics，CLEM)的活细胞成像技术，阐明Hsc70-Trio蛋白复合体在生长锥体的分子结构。 这些研究将为理解组装在神经元生长锥前沿的Trio蛋白复合体的分子功能提供结构基础。这项提案的跨学科性质将使学生有机会学习广泛的分子和细胞技术，以分析神经发育中的蛋白质复合体。目前的建议符合我的计划的长期目标，即增加对发育过程中依赖细胞骨架的基本机制的了解。

During development, the pathfinding of neurons is governed by guidance cues, such as the secreted protein netrin-1. Binding of netrin-1 to the receptor Deleted in Colorectal Cancer (DCC) expressed at the axon's distal tip, the neuronal growth cone, initiates molecular signals that modulate and change the cell cytoskeleton dynamics, leading to axon outgrowth and guidance. We identified the guanine nucleotide exchange factor (GEF) Trio as a key component of netrin-1/DCC signaling in growth cone guidance. Trio is a large (330kDa) cytoplasmic protein containing three catalytic domains, including a serine/threonine kinase domain and two GEFD1 and GEFD2 acting as activators of Rac1 and RhoA, respectively. Trio contains other motifs, including a Sec14 lipid-binding domain, eight spectrin repeats, two Src Homology 3 domains, and an immunoglobulin-like domain. The presence of these various domains suggests that Trio functions as an integrator of multiple upstream inputs and an activator of multiple downstream pathways. However, understanding how Trio integrates these signals is limited by the lack of knowledge in the structure of the protein. To characterize the mechanisms governing Trio localization and activation downstream of netrin-1/DCC, we employed a proteomic approach and identified the molecular chaperone Hsc70 as a novel Trio regulator in the developing cortex. In addition, we identified four Trio phosphorylated serine residues for which the exact function still remains to be elucidated. Overall, our recent work highlighted an important role for Trio in neurodevelopment. However, the molecular mechanisms by which Trio modulates the response of axons to guidance cues still remain elusive. Therefore, the goal of this research program is to investigate the regulation, localization, and molecular structure of Trio protein complexes at the neuronal growth cone, in three specific aims: Aim 1: To characterize the role of serine phosphorylation in Trio function and regulation Aim 2: To identify the global Trio interactome in neurite outgrowth using a BioID approach Aim 3. To elucidate the molecular structure of the Hsc70-Trio protein complex at growth cones using high-resolution molecular cryo-electron microscopy (cryo-EM) combined with live cell imaging using fluorescence microscopy (correlative light electron microscopy or CLEM). These studies will provide the structural basis for understanding the molecular functions of Trio protein complexes assembled at the leading edge of neuronal growth cones. The interdisciplinary nature of this proposal will give the students the opportunity of learning a wide range of molecular and cellular techniques to analyze protein complexes in neurodevelopment. The current proposal fits within the long-term goals of my program, which are to increase the knowledge of fundamental cytoskeletal-dependent mechanisms during development.