ROLE OF MICROTUBULE-BASED TRANSPORT IN NEURONAL POLARITY

基于微管的运输在神经元极性中的作用

• 批准号: 8416460
• 负责人: JILL C WILDONGER
• 金额: $ 24.9万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8416460
• 关键词: Acetylation; Address; Affect; Affinity; Alleles; Animal Model; Award; Axon; Behavior; Binding; Biological Assay; California; Cell Polarity; Complex; Cues; Cytoskeleton; Defect; Dendrites; Development; Distal; Drosophila genus; Dynein ATPase; Engineering; Gene Mutation; Genes; Genetic; Genomics; Goals; Human; In Vitro; Kinesin; Knock-in Mouse; Lead; Learning; Link; Mediating; Mentors; Microtubules; Miller-Dieker Syndrome; Modeling; Modification; Molecular; Molecular Motors; Morphogenesis; Motor; Motor Activity; Mus; Mutation; Nervous system structure; Neurites; Neurodevelopmental Disorder; Neuronal Migration Disorder; Neurons; Organelles; Organism; Patients; Phase; Post-Translational Protein Processing; Process; Proteins; Reagent; Research Personnel; Role; San Francisco; Shapes; Signal Transduction; Site-Directed Mutagenesis; Specific qualifier value; Specificity; Staging; Structure; System; Techniques; Testing; Tubulin; Universities; Work; Yang; base; cell motility; cofactor; developmental disease; fly; human disease; in vivo; insight; migration; neurodevelopment; neuron development; neuron loss; novel; research study; single molecule; tool

Shortly after differentiating, neurons establish distinct axonal and dendritic compartments that are specialized to send and receive signals, respectively. Polarity is essential for neurons to function in a neuronal circuit, yet how neurons polarize within a developing organism remains virtually unknown. My long-term goal as an independent biomedical researcher is to identify the mechanisms that create distinct axonal and dendritic compartments within neurons and to understand how this contributes to normal neuronal function in vivo. This proposal is based on our finding in fruit flies that the microtubule-based molecular motor dynein is necessary for two key features of neuronal polarity: the polarized localization of dendritic proteins and organelles and the uniform plus-end distal orientation of axonal microtubules. Two outstanding questions I will address in this proposal are: (1) How is dynein's function in neurons controlled by its interactions with different cofactors? and (2) How is dynein's activity regulated by its interaction with microtubules; more specifically, do microtubule modifications (such as acetylation, detyrosination, and polyglutamylation) provide spatial cues that influence dynein's activity and thereby shape neuronal polarity? The mentored phase of this award will be carried out at the University of California, San Francisco (UCSF), under the guidance of Dr. Yuh Nung Jan. During the mentored phase, I will use a genetic approach to characterize the cofactors that provide functional specificity to dynein developing fruit fly nervous system (Aim 1). Next, I will extend my studies in vitro and develop a new dynein motor construct to determine how dynein motor activity is affected by microtubule modifications (Aim 2). To do so, I will collaborate with Dr. Ronald Vale (UCSF) to learn in vitro techniques to analyze motor- microtubule interactions, including single molecule motility assays. To address how microtubule modifications affect neuronal polarization in vivo (Aim 3), I will use new knock-in technique called "genomic engineering" to build reagents for my independent phase. Dr. Yang Hong (University of Pittsburgh), who pioneered the genomic engineering technique, will serve as a consultant, as will Dr. Anthony Wynshaw-Boris (UCSF), a leader in the study of genes linked to human neurodevelopmental disorders such as classical lissencephaly. During the independent phase, I will address the following questions: Are microtubule modifications necessary for neurons to form distinct axonal and dendritic compartments in vivo? Is any one modification particularly important, or are there combinations of modifications that specify axon or dendrite formation? How do microtubule modifications regulate polarized transport in developing neurons in vivo? To answer these questions, I will use genomic engineering to knock-in multiple tubulin alleles with targeted mutations that block different microtubule modifications, both singly and in combination. Using currently available reagents and new polarity markers that I will generate, I will then characterize the effect of these mutations on neuronal polarity and dynein-mediated polarized transport within developing fruit fly nervous system. Through this combination of in vitro and in vivo approaches, these studies will provide significant new insight into microtubule-based mechanisms that shape neuronal polarity in a developing organism.

分化后不久，神经元建立了不同的轴突和树突区，这些区是特化的 分别发送和接收信号。极性对神经元在神经元回路中发挥作用是必不可少的，但 在发育中的有机体中，神经元是如何极化的几乎仍然是未知的。我的长期目标是 独立的生物医学研究人员将确定产生不同轴突和树突的机制 目的是了解神经元内部的隔室，并了解这如何有助于体内正常的神经元功能。这 这一建议是基于我们在果蝇中的发现，即基于微管的分子马达动力蛋白是必要的 对于神经元极性的两个关键特征：树突状蛋白和细胞器的极化定位以及 轴突微管远端轴突方向均匀。我将在这篇文章中解决两个悬而未决的问题 建议有：(1)动力蛋白在神经元中的功能是如何受其与不同辅因子的相互作用控制的？和 (2)动力蛋白的活性是如何通过与微管的相互作用来调节的；更具体地说，是微管 修饰(如乙酰化、去酪氨酸化和多谷氨酰化)提供空间线索，影响 动力蛋白的活性，从而形成神经元的极性？该奖项的指导阶段将在 加州大学旧金山分校(加州大学旧金山分校)在Yeh Nung博士的指导下 在指导阶段，我将使用遗传学方法来表征提供功能特异性的辅因子 动力蛋白发育果蝇神经系统(目标1)。接下来，我将扩展我的体外研究，并开发一种新的 动力蛋白马达构建以确定微管修饰如何影响动力蛋白马达活动(目标2)。 为此，我将与加州大学罗纳德·维尔博士合作，学习分析运动的体外技术。 微管相互作用，包括单分子运动性分析。为了解决微管修饰如何 影响活体神经元极化(目标3)，我将使用一种新的基因工程技术来 为我的独立阶段制作试剂。杨红博士(匹兹堡大学)，他是 基因组工程技术将担任顾问，安东尼·温肖-鲍里斯博士(加州大学旧金山分校)将担任顾问 在研究与人类神经发育障碍有关的基因方面处于领先地位，例如经典的无脑畸形。 在独立阶段，我将回答以下问题：微管是否需要修改 让神经元在活体内形成不同的轴突和树突室？有什么特别的修改吗？ 重要的是，是否有指定轴突或树突形成的修饰组合？做什么 微管修饰调节体内发育中神经元的极化运输？要回答这些问题 问题，我将使用基因组工程来敲入具有靶向突变的多个微管蛋白等位基因，从而阻止 不同的微管修饰，包括单独的和组合的。使用当前可用的试剂和新的 我将生成的极性标记，然后我将表征这些突变对神经元极性的影响 以及动力蛋白在发育中的果蝇神经系统内的极化运输。通过这种组合 这些研究将为以微管为基础的研究提供重要的新见解。 在发育中的有机体中塑造神经元极性的机制。