Role of microtubule-based transport in neuronal polarity

描述（由申请人提供）：分化后不久，神经元建立不同的轴突和树突区室，分别专门发送和接收信号。极性对于神经元在神经元回路中发挥作用至关重要，但神经元在发育中的生物体中如何运作仍然是未知的。作为一名独立的生物医学研究人员，我的长期目标是确定在神经元内创建不同轴突和树突隔室的机制，并了解这如何有助于体内正常的神经元功能。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神经元极性的两个关键特征所必需的：树突状蛋白和细胞器的极化定位和轴突微管的均匀正端远端取向。在这个提议中，我将解决两个突出的问题：（1）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？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，微管的修饰（如乙酰化、去酪氨酸化和多聚谷氨酰胺化）是否提供了影响动力蛋白活性的空间线索，从而塑造神经元的极性？该奖项的指导阶段将在加州大学旧金山分校弗朗西斯科（UCSF）进行，在Yuh Nung Jan博士的指导下。在指导阶段，我将使用遗传学方法来表征为动力蛋白发育果蝇神经系统提供功能特异性的辅因子（目标1）。接下来，我将在体外扩展我的研究，并开发一种新的动力蛋白运动结构，以确定微管修饰如何影响动力蛋白运动活性（目标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-微管相互作用，包括单分子运动测定。为了解决微管修饰如何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”，以建立我的独立阶段的试剂。基因组工程技术的先驱杨宏博士（匹兹堡大学）将担任顾问，安东尼·温肖-鲍里斯博士（加州大学旧金山分校）也将担任顾问，他是与人类神经发育障碍（如经典无脑畸形）有关的基因研究的领导者。在独立的阶段，我将解决以下问题：微管修饰必要的神经元在体内形成不同的轴突和树突隔室？是否有任何一种修饰特别重要，或者是否有特定轴突或树突形成的修饰组合？微管修饰如何调节体内发育中神经元的极化转运？为了回答这些问题，我将使用基因组工程敲入多个微管蛋白等位基因，这些等位基因具有靶向突变，可以单独或联合阻断不同的微管修饰。使用目前可用的试剂和新的极性标记，我将产生，然后我将表征这些突变对神经元极性和动力蛋白介导的极化运输在发展中的果蝇神经系统的影响。通过这种体外和体内方法的结合，这些研究将为在发育中的生物体中形成神经元极性的基于微管的机制提供重要的新见解。 公共卫生相关性：项目说明：神经元极性对于发育中的神经元正确整合到功能性神经元回路中至关重要。在发育的早期阶段，极性的丧失与几种人类发育障碍有关，包括经典的无脑回畸形。通过表征在体内控制神经元极化的基于微管的机制，该项目将为神经元如何正常极化以及如何破坏这一过程导致人类疾病提供重要的新见解。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: Project narrative: Neuronal polarity is essential for developing neurons to properly integrate into functional neuronal circuits. Loss of polarity during early stages of development has been associated with several human developmental disorders, including classical lissencephaly. By characterizing the microtubule-based mechanisms that govern neuronal polarization in vivo, this project will provide important new insight into how neurons normally polarize and how disrupting this process leads to human disease.