TRIM9 coordinates membrane trafficking and cytoskeletal dynamics

描述(由申请人提供)：成人中枢神经系统(CNS)内的连接因损伤或疾病造成的损害通常是无法修复的。要设计修复中枢神经系统损伤的治疗方法，需要详细了解中枢神经系统发育的细胞机制。随着大脑的发育，神经营养信号，如netrin，将轴突引导到其突触后目标，并诱导轴突分支以增加突触能力。无论是轴突生长锥的引导运动，还是它们的多个轴突分支，都需要相同的基本细胞机制。F-肌动蛋白和微管(MT)动力学启动和控制膜突起。胞吐作用提供磷脂和膜蛋白，为扩张的质膜提供物质。细胞骨架动力学和囊泡运输的协调可能在轴突引导和分支中发挥关键作用。然而，在轴突引导和轴突分支过程中调节这种相互作用的分子机制尚不清楚。我们的发现将TRIM9置于Netrin/DCC信号与细胞骨架和囊泡运输机制的交界处。利用小鼠遗传学、原代细胞培养、活细胞成像和神经解剖学研究相结合的方法，我的实验室发现，TRIM9缺陷的皮质神经元表现出错误的胞吐以及肌动蛋白和MT动力学缺陷。此外，我们发现缺乏TRIM9的皮质神经元存在结构性分支缺陷，不能从分支形成对netrin的响应，并且在基于netrin的轴突引导方面存在缺陷。在体内，我们发现TRIM9的缺失与皮质轴突纤维束的缺陷有关。我们的发现是TRIM9与胞外tSNARE，SNAP25相互作用和调节，这使我们假设TRIM9在空间和时间上调节生长轴突的胞吐作用。与多种细胞骨架调节因子的相互作用，包括EnA/Vasp蛋白、LamelLipodin和MAP1B，使我们假设TRIM9参与了在F-肌动蛋白和MTS动力学中发挥关键作用的蛋白质网络。由于我们已经发现TRIM9直接与Netrin受体DCC结合，并且是轴突导向线索Netrin下游所必需的，我们假设TRIM9对于细胞骨架和胞吐的协调活动是必不可少的，这些活动决定了轴突的分支和对Netrin/DCC的引导。我的实验室处于一个独特的位置，可以通过活细胞成像、定量图像分析、生物化学和小鼠模型来确定将指导信号与细胞骨架动力学和轴突分支的局部变化联系起来的分子机制。我们的长期目标是了解神经元如何整合环境线索，以协调其形态和运动的变化，这是建立功能神经系统所必需的。更好地理解轴突引导和轴突分支的机制基础，将为神经系统中的连接是如何建立的，以及它们在可塑性过程中如何重塑提供了基本的见解。我们的研究计划的结果应该对开发治疗方法以修复疾病或损伤后的这些连接具有重要价值。

DESCRIPTION (provided by applicant): Damage to connections within the adult Central Nervous System (CNS) by injury or disease is often irreparable. To design therapies to repair CNS damage requires a detailed understanding of the cellular mechanisms underlying CNS development. As the brain develops, neurotrophic cues, such as netrin, guide axons to their postsynaptic targets and induce axon branching to increase synaptic capacity. Both the guided locomotion of axonal growth cones, and their ramification into multiple axon branches, require the same fundamental cellular machinery. F-actin and microtubule (MT) dynamics initiate and steer membrane protrusions. Exocytosis delivers phospholipids and membrane proteins required to supply material to the expanding plasma membrane. Coordination of cytoskeletal dynamics and vesicle trafficking likely plays key roles in axon guidance and branching. However, the molecular mechanisms that mediate such interactions during axon guidance and axon branching are not understood. Our findings place TRIM9 at the junction of netrin/DCC signaling to both the cytoskeletal and vesicle trafficking machinery. Using a combination of mouse genetics, primary cell culture, live cell imaging and neuroanatomical studies, my lab found that TRIM9-deficient cortical neurons show misregulated exocytosis and defective actin and MT dynamics. Furthermore, we found that cortical neurons devoid of TRIM9 have constitutive branching defects, fail to form from branches in response to netrin, and are defective in netrin-based axon guidance. In vivo, we have found that loss of TRIM9 is associated with defective cortical axon fiber tracts. Our findings that TRIM9 interacts with and regulates the exocytic tSNARE, SNAP25, lead us to hypothesis that TRIM9 spatially and temporally regulates exocytosis in the growing axon. Novel interactions identified with multiple cytoskeletal regulators, including Ena/VASP proteins, Lamellipodin, and MAP1B lead us to hypothesize that TRIM9 participates in protein networks that play key roles in F-actin and MTs dynamics. As we have found that TRIM9 binds directly to the netrin receptor, DCC, and is required for functions downstream of the axon guidance cue netrin, we hypothesize that TRIM9 is essential for the coordinated activities of the cytoskeleton and exocytosis that dictate axon branching and guidance in response to netrin/DCC. My lab is in a unique position to determine the molecular mechanism that link guidance cues to local changes in cytoskeletal dynamics and axon branching through live-cell imaging, quantitative image analysis, biochemistry, and mouse models. Our long-term goal is to understand how neurons integrate environmental cues to orchestrate changes in their morphology and movement necessary to establish a functional nervous system. A better understanding of the mechanistic basis of axon guidance and axon branching will provide fundamental insight into how connections in the nervous system are established and how they are remodeled during plasticity. The results of our research plan should be of great value to the development of therapeutic approaches to repair these connections subsequent to disease or injury.