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（由申请人提供）：我在本科、学士后、研究生和博士后培训期间的研究活动和努力强烈表明了我对神经科学研究事业的承诺。我的近期目标是获得足够的支持，以促进我努力开展 K22 职业发展奖中概述的研究，并培养成功建立独立研究项目所需的技能。为了实现建立和指导自己的研究实验室的长期目标，我组建了一个导师团队，他们致力于我的职业发展和研究成功。我与主要导师 Tatyana Svitkina 博士密切合作，设计了一份职业发展计划 (CDP)，该计划补充了我之前的培训，并将帮助我成功追求我的长期职业目标。具体而言，CDP 涉及的培训活动旨在： (1) 增强批判性思维和创造性解决问题的能力； (2) 促进有效的论文并授予写作技巧； (3) 磨练口头表达技巧，促进同事间的协作互动； (4) 提升战略规划和实验室管理技能； (5) 教导科学独立性； (6) 提高指导和教学能力； (7) 教授适当的生物学原理和技术，探索在神经元中构建专门的膜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每年与我会面，讨论我在完成提案研究策略部分中概述的拟议研究目标方面取得的进展，并展示 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系（我目前正在那里接受博士后研究员的培训）非常致力于年轻科学家的研究、教育和培训。该系以及宾夕法尼亚大学的其他学校和中心组织各种职业促进活动，例如专业培训研讨会、研究研讨会、期刊俱乐部以及学生/博士后教学培训。此外，该系与宾夕法尼亚大学其他学校和中心在地理上的接近促进了协作环境，并使我能够使用许多研究设施，这些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。在理解轴突起始段（AIS）如何调节动作电位启动和维持神经元极性方面存在根本性的差距，部分原因是对其分子结构和关键细胞骨架成分、锚蛋白、βIV-血影蛋白和肌动蛋白丝的贡献了解甚少。这一差距的持续存在代表了一个重要的问题，因为在它被填补之前，理解由于神经元损伤或 AIS 蛋白突变而导致 AIS 破坏而发生的神经系统疾病在很大程度上仍然是不可理解的。本应用的目的是确定构成 AIS 细胞骨架核心框架的基本 AIS 组件的相对排列，并定义 βIV-血影蛋白的特定作用。中心假设是 βIV-血影蛋白、锚蛋白和肌动蛋白丝在 AIS 涂层内形成广泛的、互连的网络，有助于 AIS 膜的特性和/或功能。我提出的研究的基本原理是，一旦了解单个 AIS 成分如何影响 AIS 细胞骨架结构和功能，就会更好地理解 AIS 以及可能的神经元极性如何在神经元损伤或疾病的情况下得以保留或恢复。为了检验中心假设并实现本提案的目标，将追求以下具体目标：1）确定关键AIS外壳细胞骨架成分、βIV-血影蛋白、锚蛋白G和肌动蛋白丝的空间排列（当前研究，第一阶段）； 2) 确定 βIV-血影蛋白在 AIS 细胞骨架结构和功能中的作用（第二阶段）。该假设将通过基因操作技术（RNAi、突变基因和显性失活构建体）进行测试，以去除不属于核心锚蛋白、βIV-血影蛋白和肌动蛋白丝细胞骨架的 AIS 蛋白。这将生成 AIS 的简化版本，有助于使用铂复制电子显微镜 (PREM)、immunoPREM 和电子断层扫描进行详细的结构分析。该提案中的研究具有创新性，因为它采用了组合方法，代表了解决基本 AIS 细胞骨架结构的新方法，提出了新假设，因此可以获取新知识。这项拟议的研究意义重大，因为它有望纵向推进和扩大对 AIS 分子组织破坏如何导致不同神经系统疾病的理解。一旦获得这些知识，预计将促进我们对神经系统损伤后如何恢复或保留 AIS 的理解，这将保留神经元的极性和产生动作电位的能力。此外，可以预期更好地了解膜细胞骨架如何在其他基于锚蛋白-血影蛋白的膜域（例如，Ranvier节点、无髓鞘轴突、心肌细胞T管和闰盘、上皮侧膜、顾客和光感受器内/外节段）中组织。