Axon initial segment made simple: Architecture of the cytoskeletal network

 描述（由申请人提供）：我的研究活动和努力，在我的本科，学士后，研究生和博士后培训是我致力于在神经科学研究的职业生涯的强烈迹象。我的近期目标是获得足够的支持，这将有助于我努力开展本K22职业发展奖中概述的研究，并发展必要的技能，成功地建立一个独立的研究计划。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Tatyana Svitkina博士密切合作，设计了一个职业发展计划（CDP），补充了我之前的培训，并将帮助我成功地追求我的长期职业目标。具体而言，CDP涉及的培训活动旨在：（1）提高批判性思维和创造性解决问题的技能;（2）促进有效的论文和赠款写作技能;（3）磨练口头陈述技能，促进学院和合作互动;（4）促进战略规划和实验室管理技能;（5）培养科学独立性;（6）提高辅导和教学技能;（7）提高学生的专业技能;（8）提高学生的专业技能;（9）提高学生的专业技能;（10）提高学生的专业技能;（11）提高学生的专业技能;（12）提高学生的专业技能;（13）提高学生的专业技能;（14）提高学生的专业技能;（15）提高学生的专业技能;（16）提高学生的专业技能;（17）提高学生的专业技能;（18）提高学生的专业技能;（19（7）教授适当的生物学原理和技术，以探索在神经元中构建专门的膜结构域的分子和细胞骨架机制。为了确保CDP达到预期目的，导师团队/咨询委员会将每月和每年与我会面，讨论我在完成提案的研究策略部分中概述的拟议研究目标方面的进展情况，并展示CDP中概述的价值观和技能。 环境宾夕法尼亚大学（Penn）和宾夕法尼亚大学艺术与科学学院的生物系，我目前正在接受博士后研究员的培训，都致力于研究，教育和培训年轻科学家。该部门，以及其他学校和中心在宾夕法尼亚大学，组织各种职业促进活动，如专业培训讲习班，研究研讨会，期刊俱乐部，和学生/博士后培训教学。此外，该部门与宾夕法尼亚大学其他学校和中心的地理位置接近，为协作环境提供了便利，并使我能够访问大量研究设施，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。 RESEARCH.在理解轴突起始段（AIS）如何调节动作电位起始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组分（ankrexG、β IV-血影蛋白和肌动蛋白丝）的贡献的理解不足。这一差距的持续存在代表了一个重要的问题，因为在填补这一差距之前，理解由于AIS破坏（通过神经元损伤或AIS蛋白突变）而发生的神经系统疾病在很大程度上仍然是不可理解的。本申请的目的是确定形成AIS细胞骨架核心框架的基本AIS组分的相对排列，并定义β IV-血影蛋白的特定作用。中心假设是β IV-血影蛋白、锚蛋白G和肌动蛋白丝在AIS被膜内形成广泛的相互连接的网络，其有助于AIS膜的性质和/或功能。我提出的研究的基本原理是，一旦知道了个别AIS组分如何有助于AIS细胞骨架结构和功能，就可以更好地理解AIS以及可能的神经元极性如何在神经元损伤或疾病的背景下得以保留或恢复。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外壳细胞骨架组分β IV-血影蛋白、锚蛋白G和肌动蛋白丝的空间排列（当前研究，I期）; 2）确定β IV-血影蛋白在AIS细胞骨架结构和功能中的作用（II期）。将通过遗传操作技术（RNAi、突变基因和显性阴性构建体）来检测这一假设，以去除不属于核心锚蛋白G、βIV-血影蛋白和肌动蛋白丝细胞骨架一部分的AIS蛋白。这将产生AIS的简化版本，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。该提案中的研究是创新的，因为它采用了一种组合方法，代表了一种解决基本AIS细胞骨架结构的新方法，解决了新的假设，因此可以获得新的知识。这项拟议的研究意义重大，因为它有望垂直推进和扩大对AIS分子组织破坏如何导致不同神经系统疾病的理解。一旦这些知识可用，预计将促进我们对AIS如何在神经系统损伤后恢复或保留的理解，这将保留神经元的极性和产生动作电位的能力。此外，更好地基本理解膜细胞骨架如何在其他基于锚蛋白-血影蛋白的膜结构域（例如，Ranvier结节、无髓轴突、心肌细胞T-小管和闰盘、上皮细胞侧膜、细胞外膜和光感受器内/外节）。

 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.