Role of microtubule-based transport in neuronal polarity

描述(申请人提供)：分化后不久，神经元建立不同的轴突和树突间隔，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是必不可少的，然而在发育中的有机体中神经元是如何极化的实际上仍然是未知的。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中创建不同轴突和树突间隔的机制，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Dynein是神经元极性的两个关键特征所必需的：树突蛋白和细胞器的极化定位和轴突微管的均匀正端远端定向。我将在这项提案中解决的两个突出问题是：(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？以及(2)动力蛋白的活性是如何通过它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、去酪氨酸化和多谷氨酰化)是否提供了影响动力蛋白活性从而塑造神经元极性的空间线索？该奖项的指导阶段将在加州大学旧金山分校(UCSF)进行，由Yeh Nung Jan.博士指导。在指导阶段，我将使用遗传学方法来表征为动力蛋白发育中的果蝇神经系统提供功能特异性的辅助因素(目标1)。接下来，我将扩展我的体外研究，开发一种新的动力蛋白马达结构，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-微管相互作用的体外技术，包括单分子运动性分析。为了解决微管修饰如何影响体内神经细胞极化的问题(目标3)，我将使用被称为“基因组工程”的新敲入技术来为我的独立阶段构建试剂。基因组工程技术的先驱杨鸿博士(匹兹堡大学)将担任顾问，加州大学旧金山分校(UCSF)的安东尼·温肖-鲍里斯博士(Anthony Wynshaw-Boris)将担任顾问，他是与经典小脑等人类神经发育障碍相关的基因研究的领先者。在独立阶段，我将回答以下问题：在活体中，神经元形成不同的轴突和树突室是否需要微管的修改？有没有哪一种修饰特别重要，或者有没有指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运输？为了回答这些问题，我将使用基因组工程来敲击具有靶向突变的多个微管蛋白等位基因，这些突变可以阻止不同的微管修饰，包括单独的和组合的。使用目前可用的试剂和我将产生的新的极性标记，然后我将表征这些突变对发育中的果蝇神经系统内神经元极性和动力蛋白介导的极化运输的影响。通过这种体外和体内方法的结合，这些研究将为基于微管的机制提供重要的新见解，这些机制在发育中的有机体中塑造神经元的极性。 公共卫生相关性：项目叙述：神经元的极性对于发育中的神经元正确地融入功能神经元回路是必不可少的。在发育的早期阶段，极性的丧失与一些人类发育障碍有关，包括经典的无脑畸形。通过表征体内控制神经元极化的基于微管的机制，该项目将为神经元正常极化以及破坏这一过程如何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.