Axon initial segment made simple: Architecture of the cytoskeletal network

轴突初始段变得简单：细胞骨架网络的架构

• 批准号: 9033341
• 负责人: Steven L Jones
• 金额: $ 9.85万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-30 至 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9033341
• 关键词: Actins; Action Potentials; Address; Advisory Committees; Ankyrins; Architecture; Arts; Axon; Biological; Biology; Blood Cells; Cardiac Myocytes; Cell Adhesion Molecules; Complement; Complex; Critical Thinking; Cytoskeletal Proteins; Cytoskeleton; Data; Dendrites; Development Plans; Diffusion; Disease; Dominant-Negative Mutation; Educational process of instructing; Educational workshop; Electron Microscopy; Ensure; Epilepsy; Epithelial; Exhibits; Family member; Fire - disasters; Gated Ion Channel; Genes; Goals; Grant; Individual; Intercalated disc; Ion Channel; Journals; K-Series Research Career Programs; Knowledge; Laboratories; Laboratory Research; Lateral; Lead; Life; Lipid Bilayers; Maintenance; Mechanics; Membrane; Mentors; Microfilaments; Mission; Molecular; Muscle Cells; Mutation; National Institute of Neurological Disorders and Stroke; Nervous System Physiology; Nervous System Trauma; Neurologic; Neuronal Injury; Neurons; Neurosciences Research; Oral; Paper; Pathogenesis; Pennsylvania; Phase; Photoreceptors; Platinum; Play; Prevention; Problem Solving; Property; Proteins; Public Health; RNA Interference; Ranvier' s Nodes; Relative (related person); Research; Research Activity; Research Personnel; Resources; Role; Scaffolding Protein; Schizophrenia; Schools; Science; Scientist; Spectrin; Strategic Planning; Structure; Students; Techniques; Testing; Three-dimensional analysis; Training; Training Activity; Training and Education; Universities; Vertebral column; Work; Writing; base; betaIV spectrin; career; career development; cell type; collaborative environment; density; design; electron tomography; genetic manipulation; innovation; meetings; mutant; nervous system disorder; neural circuit; neurotransmission; post-doctoral training; prevent; programs; public health relevance; research and development; research facility; scaffold; skills; success; tool; voltage

 DESCRIPTION (provided by applicant): My research activities and efforts during my undergraduate, post-baccalaureate, graduate, and postdoctoral training are strong indications of my commitment to a career in neuroscience research. My immediate goal is to acquire sufficient support that will facilitate my endeavors to carry out the studies outlined in this K22 Career Development Award and develop the necessary skills to successfully establish an independent research program. To reach my long term goal of establishing and directing my own research laboratory, I have assembled a team of mentors who are committed to my career development and research success. I have worked closely with the primary mentor, Dr. Tatyana Svitkina, to design a Career Development Plan (CDP) that complements my prior training and will aid me in the successful pursuit of my long-term career goal. Specifically, the CDP involves training activities designed to: (1) Enhance critical thinking and creative problem solving skills; (2) Promote effective paper and grant writing skills; (3) Hone oral presentation skills and promote collegial and collaborative interactions; (4) Promote strategic planning and laboratory management skills; (5) Teach Scientific Independence; (6) Enhance mentoring and teaching skills; (7) Teach appropriate biological principles and techniques to explore the molecular and cytoskeletal mechanisms for building specialized membrane domains in neurons. To ensure that the CDP serves its intended purpose, the Mentoring Team/Advisory Committee will meet with me on a monthly and annual basis to discuss my progress toward completing the proposed research objectives outlined in the Research Strategy Section of the proposal and demonstrating the values and skills outlined in the CDP. ENVIRONMENT. The University of Pennsylvania (Penn) and the Department of Biology in Penn's School of Arts and Sciences, where I am currently being trained as a postdoctoral researcher, are strongly dedicated to research, education, and training of young scientists. The Department, as well as other schools and centers at Penn, organizes various career-promoting activities, such as professional-training workshops, research seminars, journal clubs, and student/postdoctoral training in teaching. Also, the geographical proximity of the Department to other schools and centers at Penn facilitates a collaborative environment and allows me access to a host of research facilities that will afford me the tools and resources required to successfully execute the research proposed in this application. RESEARCH. There is a fundamental gap in understanding how the axon initial segment (AIS) regulates action potential initiation and maintains neuron polarity, partly due to poor understanding of its molecular architecture and contribution of key cytoskeletal components, ankyrinG, βIV-spectrin, and actin filaments. Continued existence of this gap represents an important problem because, until it is filled, understanding neurological disorders that occur as a result of AIS disruption through neuronal injury or mutation of AIS proteins will largely remain incomprehensible. The objective of this application is to determine the relative arrangement of fundamental AIS components that form the core framework of the AIS cytoskeleton and define specific roles for βIV-spectrin. The central hypothesis is that βIV-spectrin, ankyrinG and actin filaments form an extensive, interconnected network within the AIS coat that contributes to AIS membrane properties and/or function. The rationale of my proposed research is that once it is known how individual AIS components contributes to AIS cytoskeletal structure and function, it will be better understood how the AIS, and possibly neuron polarity, might be preserved or restored in the context of neuronal injury or disease. In order to test the central hypothesis and accomplish the objective of this proposal, the following specific aims will be pursued: 1) Determine the spatial arrangement of key AIS coat cytoskeletal components, βIV-spectrin, ankyrinG, and actin filaments (current research, Phase I); and 2) Determine the role of βIV-spectrin in AIS cytoskeletal structure and function (Phase II). This hypothesis will be tested by genetic manipulation techniques (RNAi, mutant genes, and dominant-negative constructs) to remove AIS proteins that are not part of the core ankyrinG, βIV- spectrin, and actin filament cytoskeleton. This will generate a simplified version of the AIS that will facilitate detailed structural analysis using platinum replica electron microscopy (PREM), immunoPREM, and electron tomography. The research in the proposal is innovative because it employs a combinatory approach that represents a new way for resolving the basic AIS cytoskeletal architecture, addresses new hypotheses, and, therefore, allows acquisition of new knowledge. This proposed research is significant because it is expected to vertically advance and expand understanding on how disruption of the molecular organization of the AIS leads to different neurological conditions. Once such knowledge is available it is expected to promote our understanding of how the AIS can be restored or preserved following nervous system injury, which will preserve a neuron's polarity and ability to generate action potentials. Furthermore, better fundamental understanding of how the membrane cytoskeleton is organized in other ankyrin-spectrin based membrane domains (e.g., nodes of Ranvier, unmyelinated axons, cardio myocyte T-tubules and intercalated disks, epithelial lateral membranes, costumers, and photoreceptor inner/outer segments) can be anticipated.

 描述（由适用提供）：我的研究活动和在本科，学士后，毕业生和博士后培训期间的研究活动和博士后培训是我对神经科学研究事业的承诺的有力迹象。我的近期目标是获得足够的支持，以支持我努力进行这项K22职业发展奖中概述的研究，并发展成功建立独立研究计划的必要技能。为了达到建立和指导自己的研究实验室的长期目标，我组建了一个致力于我的职业发展和研究成功的导师团队。我与主要导师Tatyana Svitkina博士紧密合作，设计了一项职业发展计划（CDP），该计划完成了我先前的培训，并将帮助我成功地追求我的长期职业目标。具体而言，CDP涉及旨在：（1）增强批判性思维和创造性解决问题技能的培训活动； （2）促进有效的纸张和授予写作技巧； （3）磨练的口头表现技巧，并促进联盟和协作互动； （4）促进战略规划和实验室管理技能； （5）教授科学独立性； （6）增强心理和教学技能； （7）教授适当的生物学原理和技术来探索用于在神经元中构建专门膜结构域的分子和细胞骨架机制。为了确保CDP达到其预期目的，指导团队/咨询委员会将每月和年度与我会面，以讨论我在提案的“研究策略”部分中概述的拟议研究目标的进展，并证明CDP中概述的价值观和技能。环境。宾夕法尼亚大学（宾夕法尼亚大学）和宾夕法尼亚大学艺术与科学学院生物学系，我目前正在接受博士后研究员的培训，非常致力于对年轻科学家的研究，教育和培训。该系以及宾夕法尼亚州的其他学校和中心都组织各种职业促进活动，例如专业培训讲习班，研究中心，期刊俱乐部以及学生/博士后教学培训。此外，该部门与宾夕法尼亚州设施的其他学校和中心的地理距离是协作环境，并使我可以访问许多研究设施，这些研究设施将为我提供成功执行本申请中提出的研究所需的工具和资源。研究。理解轴突初始段（AIS）如何调节动作潜在计划并保持神经元极性存在根本差距，部分原因是对其分子结构的了解不足以及关键细胞骨架成分，Ankyring，aNkyring，βIV-Spectrin，β-spectrin和actin丝蛋白丝的贡献。持续存在此差距是一个重要的问题，因为在填补之前，了解由于AIS通过神经元损伤或AIS蛋白突变而导致的神经系统疾病将在很大程度上仍然无法理解。该应用的目的是确定构成AIS细胞骨架的核心框架的基本AIS组件的相对排列，并定义β-spectrin的特定作用。中心假设是β-spectrin，Ankyring和肌动蛋白丝形成了AIS涂层中广泛的，相互连接的网络，有助于AIS膜性质和/或功能。我提出的研究的理由是，一旦知道单个AIS成分如何促进AIS细胞骨架结构和功能，就可以更好地理解AIS和可能的神经元极性如何在神经元损伤或疾病的背景下保留或恢复。为了检验中心假设并实现该提案的目标，将追求以下特定目标：1）确定关键AIS涂层细胞骨架成分的空间排列，β-spectrin，Ankyring和肌动蛋白丝（当前的研究，I阶段I）； 2）确定该作用将生成AIS的简化版本，该版本将促进使用Platinum Replica Electron显微镜（PER），Immunoprem和电子断层扫描的详细结构分析。该提案中的研究具有创新性，因为它采用了一种组合方法，它代表了解决基本AIS细胞骨架架构的新方法，解决了新的假设，因此可以获取新知识。这项提出的研究很重要，因为它有望垂直提高和扩展对AIS分子组织的破坏如何导致不同神经系统疾病的理解。一旦获得此类知识，预计将在神经系统损伤后促进我们对AIS如何恢复或保存的理解，这将保持神经元的极性和产生动作电位的能力。此外，对膜细胞骨架如何在其他基于Ankyrin-折线蛋白的膜结构域（例如Ranvier的淋巴结，无髓囊轴突，有氧运动肌细胞T-Tubulles和Eferceptrane coptranes codgert cogemerecemers和cotgector）中/外层。