Actin-Based Vesicle Transport in Neurons

神经元中基于肌动蛋白的囊泡运输

• 批准号: 6542630
• 负责人: George M. Langford
• 金额: $ 10万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-01 至 2004-08-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6542630
• 关键词: SDS polyacrylamide gel electrophoresis; actins; affinity chromatography; axon; biological transport; crosslink; density gradient ultracentrifugation; endoplasmic reticulum; fluorescence resonance energy transfer; immunoprecipitation; kinesin; mass spectrometry; microfilaments; microtubules; molecular cloning; myosins; neurofilament; neurons; protein binding; protein protein interaction; protein sequence; squid; vesicle /vacuole; western blottings; SDS polyacrylamide gel electrophoresis; actins; affinity chromatography; axon; biological transport; crosslink; density gradient ultracentrifugation; endoplasmic reticulum; fluorescence resonance energy transfer; immunoprecipitation; kinesin; mass spectrometry; microfilaments; microtubules; molecular cloning; myosins; neurofilament; neurons; protein binding; protein protein interaction; protein sequence; squid; vesicle /vacuole; western blottings

DESCRIPTION (provided by applicant):Long term potentiation (LTP) and long term depression (LTD) in developing and mature nervous systems require the release of calcium from smooth ER located at the synapse. Myosin V is postulated to play an important role in this process by transporting ER to the synapse where repetitive electrical activity triggers calcium release. Genetic studies with dilute (MyoVa null) mice have shown that mutations in myosin V lead to neurological defects including opisthotonos and ataxia. The neurological disorder in humans known as Griscelli Syndrome has been linked to mutations in the human gene for myosin V.In this research project we plan to provide additional evidence that transport of ER in the actin-rich regions of neurons is driven by myosin V. We will use the squid nervous system as a model for these studies. ER vesicles in the squid giant axon retain the ability to move on actin filaments in vitro and we have shown that myosin V is the vesicle motor. Our lab was the first to show that these vesicles move on both microtubules and actin filaments, an observation that led to the 'dual transport' model. Our principal goal is to determine the mechanism by which vesicle transport on microtubules and actin filaments is coordinated and regulated. We plan to test the hypothesis that myosin V and kinesin form a complex on vesicles through tail-tail interactions and that feedback between the proteins facilities the transition of vesicles from microtubules to actin filaments.The overall goal of this research project is to isolate, identify and characterize the myosin-V/kinesin motor complex. The first specific aim is to isolate the complex and to determine the proteins that link the complex to the surface of vesicles. The second specific aim of this project is to establish that the tail domains of myosin V and kinesin interact directly when the two motors form a complex on vesicles. The third specific aim is to determine if binding to cargo (vesicles) activates the motors and whether feedback between the tails of myosin V and kinesin alters motor activity. Myosin V complexes and vesicle fractions will be isolated from squid brain. Biochemical techniques including chemical cross-linking, affinity chromatography and immunoprecipitation will be used to identify novel proteins in the hetero-motor complex. Novel proteins will be cloned and sequenced. Co-sedimentation assays and FRET microscopy will be used to determine if feedback between the two motors in the multi-protein complex alters motor activity. These studies will advance our understanding of the mechanism by which long-range movement of vesicles on microtubules and short-range movement on actin filaments are coordinated.

描述（由申请人提供）：在发育和成熟的神经系统中，长期增强（LTP）和长期抑郁（LTD）需要从位于突触的平滑ER释放钙。假定肌球蛋白V通过将ER运输到重复的电活动触发钙释放的突触中，从而在此过程中发挥重要作用。用稀释的遗传研究（Myova Null）小鼠表明，肌球蛋白V中的突变导致神经系统缺陷，包括粘具和共济失调。人类的神经系统疾病被称为肌球蛋白V的人类基因中的突变有关，我们计划提供其他证据，表明肌球蛋白V驱动ER在富含肌动蛋白的神经元中的运输是由肌球蛋白V驱动的。我们将使用Squid神经系统作为这些研究的模型。鱿鱼巨型轴突中的ER囊泡保留在体外移动肌动蛋白丝的能力，我们已经表明肌球蛋白V是囊泡运动。我们的实验室是第一个表明这些囊泡在微管和肌动蛋白丝上移动的，这一观察结果导致了“双转运”模型。我们的主要目标是确定微管和肌动蛋白丝上的囊泡运输的机制。我们计划通过尾尾相互作用以及蛋白质设施之间的反馈在囊泡上形成复合物的假设，即囊泡从微管到肌动蛋白丝的过渡。该研究项目的总体目标是隔离，鉴定并鉴定并鉴定并表征肌动蛋白-V/动蛋白运动蛋白运动蛋白运动蛋白运动复合物。第一个具体目的是隔离复合物并确定将复合物与囊泡表面联系起来的蛋白质。该项目的第二个具体目的是确定当两个电动机在囊泡上形成复合物时，肌球蛋白V和驱动蛋白的尾部结构域直接相互作用。第三个具体目的是确定与货物（囊泡）结合是否激活电动机，以及肌球蛋白V和驱动蛋白尾部之间的反馈是否会改变运动活性。肌球蛋白V复合物和囊泡分数将从鱿鱼大脑中分离出来。包括化学交联，亲和力色谱和免疫沉淀在内的生化技术将用于鉴定杂体复合物中的新蛋白质。新型蛋白质将被克隆和测序。共进行测定测定和FRET显微镜将用于确定多蛋白复合物中两个电动机之间的反馈是否改变了运动活动。这些研究将促进我们对囊泡在微管上的远距离运动和肌动蛋白丝上短距离运动的理解。