ROLE OF MICROTUBULE-BASED TRANSPORT IN NEURONAL POLARITY

在分化后不久，神经元建立不同的轴突和树突区室， 以分别发送和接收信号。极性对于神经元在神经元回路中的功能是必不可少的，然而 神经元在发育中的有机体中是如何生长的仍然是个未知数。我的长期目标是 一个独立的生物医学研究人员是确定机制，创造不同的轴突和树突 我们的目的是了解神经元内的隔室，并了解这如何有助于体内正常的神经元功能。这 我们在果蝇中发现，基于微管的分子动力蛋白是必需的， 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， 轴突微管的均匀正端远端取向。我将在这篇文章中讨论两个悬而未决的问题。 动力蛋白在神经元中的功能是如何通过与不同辅因子的相互作用来控制的？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，微管 修饰（如乙酰化、脱酪氨酸和多聚谷氨酰胺化）提供了影响 动力蛋白的活动从而形成神经元极性？该奖项的指导阶段将在 加州大学旧金山分校弗朗西斯科（UCSF），在博士的指导下。 在指导阶段，我将使用遗传方法来表征提供功能特异性的辅助因子， 动力蛋白发育果蝇神经系统（Aim 1）。接下来，我将在体外扩展我的研究， 动力蛋白运动结构，以确定微管修饰如何影响动力蛋白运动活性（目的2）。 为此，我将与罗纳德瓦尔博士（加州大学旧金山分校）合作，学习体外技术来分析运动神经元。 微管相互作用，包括单分子运动测定。为了解决微管修饰 影响体内神经元极化（目标3），我将使用新的敲入技术称为"基因组工程"， 为我的独立阶段制造试剂杨宏博士（匹兹堡大学），谁开创了 基因组工程技术，将担任顾问，博士安东尼温肖鲍里斯（加州大学旧金山分校）， 他是与经典无脑畸形等人类神经发育障碍相关基因研究的领导者。 在独立阶段，我将解决以下问题：微管修饰是否必要 神经元在体内形成不同的轴突和树突区室？是否有任何一个修改特别 重要，还是有特定轴突或树突形成的修饰组合？怎么 微管修饰调节体内发育中神经元的极化运输？回答这些 问题，我将使用基因组工程敲入多个微管蛋白等位基因与靶向突变，阻止 不同的微管修饰，无论是单独的还是组合的。使用现有试剂和新的 我将生成极性标记，然后我将描述这些突变对神经元极性的影响 以及果蝇神经系统发育过程中动力蛋白介导的极化运输。通过这种结合 在体外和体内的方法，这些研究将提供重要的新的见解，微管为基础的 在发育中的有机体中形成神经元极性的机制。

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.