Axon initial segment made simple: Architecture of the cytoskeletal network

 描述(由申请人提供)：我在本科、学士、研究生和博士后培训期间的研究活动和努力是我致力于神经科学研究事业的强烈迹象。我的直接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学习，并发展必要的技能，以成功地建立一个独立的研究项目。为了实现建立和指导自己的研究实验室的长期目标，我组建了一支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Tatana Svitina博士密切合作，设计了一份职业发展计划(CDP)，该计划与我之前的培训相辅相成，将帮助我成功地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)提高批判性思维和解决问题的创造性技能；(2)促进有效的论文和补助金撰写技能；(3)磨练口头陈述技能，促进合作和协作互动；(4)促进战略规划和实验室管理技能；(5)教授科学独立；(6)加强指导和教学技能；(7)教授适当的生物学原理和技术，以探索在神经元中建立专门的膜域的分子和细胞骨架机制。为了确保CDP达到预期的目的，指导团队/咨询委员会将每月和每年与我会面，讨论我在完成提案中研究战略部分列出的拟议研究目标方面的进展情况，并展示CDP中列出的价值观和技能。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的生物系，我目前正在那里接受博士后研究培训，致力于研究、教育和培训年轻科学家。该部门以及宾夕法尼亚大学的其他学校和中心组织各种促进职业发展的活动，如专业培训讲习班、研究研讨会、期刊俱乐部和教学方面的学生/博士后培训。此外，该系距离宾夕法尼亚大学其他学校和中心的地理位置很近，这为合作环境提供了便利，并使我能够访问许多研究设施，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。轴突起始段(AIS)如何调节动作电位的启动和维持神经元的极性有一个根本的空白，部分原因是对它的分子结构和关键细胞骨架成分--骨架蛋白G、βIV-SPECTIN和肌动蛋白细丝的作用缺乏了解。这个缺口的持续存在代表着一个重要的问题，因为在这个缺口被填补之前，理解由于神经元损伤或AIS蛋白突变而导致的AIS中断所导致的神经疾病在很大程度上仍然是无法理解的。本申请的目的是确定构成人工免疫系统细胞骨架核心框架的基本人工免疫系统组件的相对排列，并定义βIV-血影蛋白的特定作用。中心假设是βIV-Spectrin、ankyrinG和肌动蛋白细丝在AIS膜内形成一个广泛的、相互连接的网络，这有助于AIS膜的特性和/或功能。我提出的研究的基本原理是，一旦了解了单个AIS组件如何对AIS细胞骨架结构和功能做出贡献，就会更好地理解AIS，可能还有神经元的极性，在神经元损伤或疾病的背景下如何保存或恢复。为了验证中心假说和实现这一建议的目标，将追求以下具体目标：1)确定关键的人工免疫系统外壳细胞骨架成分、βIV-光谱蛋白、骨架G和肌动蛋白细丝的空间排列(当前研究，第一阶段)；以及2)确定βIV-光谱蛋白在人工免疫系统细胞骨架结构和功能中的作用(第二阶段)。这一假设将通过遗传操作技术(RNA干扰、突变基因和显性负向构造)进行验证，以移除不属于核心骨架G、βIV-SPECTIN和肌动蛋白细丝细胞骨架的AIS蛋白。这将生成简化版本的AIS，这将有助于使用铂复制电子显微镜(PREM)、免疫PREM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，因为它采用了一种组合方法，代表了解决基本AIS细胞骨架结构的新方法，解决了新的假设，因此允许获得新的知识。这项拟议的研究具有重要意义，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疾病的理解。一旦有了这样的知识，它有望促进我们对神经系统损伤后如何恢复或保存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，更好地了解膜细胞骨架是如何在其他基于ankyrin-spectrin的膜域(例如，Ranvier结节、无髓轴突、心肌细胞T管和间盘、上皮侧膜、服装和光感受器内外节段)中组织的有望得到更好的基础理解。

 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.