TRIM9 coordinates membrane trafficking and cytoskeletal dynamics

TRIM9 协调膜运输和细胞骨架动力学

• 批准号: 8991497
• 负责人: Stephanie Gupton
• 金额: $ 35万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-01-01 至 2018-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8991497
• 关键词: Actins; Adult; Affect; Area; Axon; Behavior; Binding; Biochemistry; Brain; Cell membrane; Complex; Corpus Callosum; Cues; Cytoskeletal Proteins; Cytoskeleton; Data; Defect; Development; Disease; Electroporation; Embryo; Exhibits; Exocytosis; F-Actin; Fiber; Filopodia; Gap Junctions; Genetic; Goals; Growth Cones; Health; Human; Image; Image Analysis; In Vitro; Individual; Injury; Knockout Mice; Lead; Link; Locomotion; Mediating; Membrane; Membrane Protein Traffic; Membrane Proteins; Microfluidic Microchips; Microscopy; Microtubule Polymerization; Microtubules; Molecular; Morphology; Movement; Mus; Nerve; Nerve Fibers; Nervous System Trauma; Nervous system structure; Neuraxis; Neurites; Neurologic; Neurons; Phenotype; Phospholipids; Play; Positioning Attribute; Primary Cell Cultures; Protein Family; Proteins; Rehabilitation therapy; Research; SNAP receptor; Signal Transduction; Spinal cord injury; Surface; Synapses; Syndrome; Testing; Vertebral column; Vesicle; Work; axon growth; axon guidance; base; central nervous system injury; density; in utero; in vivo; insight; live cell imaging; microtubule-associated protein 1B; mouse model; mutant; nervous system development; netrin receptor; novel; polymerization; postsynaptic; quantitative imaging; receptor; relating to nervous system; repaired; response; therapeutic development; therapy design; trafficking; ubiquitin ligase; ubiquitin-protein ligase; vasodilator-stimulated phosphoprotein

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.

描述（由申请人提供）：损伤或疾病对成人中枢神经系统（CNS）内连接的损害通常是不可修复的。设计治疗方法来修复中枢神经系统损伤需要详细了解中枢神经系统发展的细胞机制。随着大脑的发育，神经营养因子（如netrin）将轴突引导到它们的突触后靶点，并诱导轴突分支以增加突触能力。轴突生长锥的引导运动和它们分化成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）动力学启动和操纵膜突起。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导向和分支中起关键作用。然而，在轴突引导和轴突分支过程中介导这种相互作用的分子机制尚不清楚。我们的研究结果将TRIM 9置于netrin/DCC信号传导到细胞骨架和囊泡运输机制的连接处。使用小鼠遗传学，原代细胞培养，活细胞成像和神经解剖学研究的组合，我的实验室发现TRIM 9缺陷的皮质神经元表现出失调的胞吐作用和缺陷的肌动蛋白和MT动力学。此外，我们发现缺乏TRIM 9的皮质神经元具有组成性分支缺陷，未能响应netrin从分支形成，并且在基于netrin的轴突引导中存在缺陷。在体内，我们发现TRIM 9的缺失与皮质轴突纤维束缺陷有关。我们的研究结果，TRIM 9相互作用，并调节胞吐tSNARE，SNAP 25，使我们的假设，TRIM 9空间和时间调节胞吐在生长的轴突。与多种细胞骨架调节剂，包括Ena/VASP蛋白，Lamelliopodin和MAP 1B确定的新的相互作用使我们假设TRIM 9参与在F-肌动蛋白和MT动力学中发挥关键作用的蛋白质网络。由于我们已经发现TRIM 9直接结合到netrin受体DCC，并且是轴突导向因子netrin下游功能所必需的，因此我们假设TRIM 9对于细胞骨架和胞吐作用的协调活动是必不可少的，所述细胞骨架和胞吐作用决定轴突分支和响应netrin/DCC的导向。我的实验室是在一个独特的位置，以确定通过活细胞成像，定量图像分析，生物化学和小鼠模型的细胞骨架动力学和轴突分支的局部变化的分子机制，链接的指导线索。我们的长期目标是了解神经元如何整合环境线索，以协调建立功能性神经系统所需的形态和运动变化。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如何建立以及它们在可塑性期间如何重塑提供基本的见解。我们的研究计划的结果应该是非常有价值的治疗方法，以修复这些连接后，疾病或损伤的发展。