Function of Microtubule Plus-End-Tracking Proteins in the Neuronal Growth Cone

神经元生长锥中微管加端追踪蛋白的功能

• 批准号: 8781246
• 负责人: Laura Anne LOWERY
• 金额: $ 24.9万
• 依托单位: BOSTON COLLEGE
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-02-01 至 2017-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8781246
• 关键词: Advisory Committees; Affect; Autistic Disorder; Automobile Driving; Axon; Behavior; Binding; Biochemical; Biological Assay; Biology; Biomedical Research; Brain; Caenorhabditis elegans; Cellular biology; Commit; Complement; Computer Analysis; Computing Methodologies; Country; Cues; Cytoskeleton; Data; Decision Making; Development; Disease; Doctor of Philosophy; Embryo; Environment; Event; Faculty; Fostering; Frequencies; Future; Genes; Genetic; Goals; Growth; Growth Cones; Image; Image Analysis; Imagery; In Vitro; Journals; Knowledge; Lead; Life; Logic; Measures; Mental disorders; Mentors; Mentorship; Methods; Microscopy; Microtubule Polymerization; Microtubules; Modeling; Morphogenesis; National Research Service Awards; Nature; Nervous System Physiology; Neurobiology; One-Step dentin bonding system; Paper; Pathway interactions; Phase; Play; Plus End of the Microtubule; Population; Positioning Attribute; Postdoctoral Fellow; Prevention; Protein Family; Protein Tyrosine Kinase; Proteins; Publications; Records; Regulation; Research; Research Personnel; Research Training; Resolution; Retinal Cone; Role; Schizophrenia; Signal Pathway; Signal Transduction; Techniques; Testing; Time; Training; Training Support; Universities; Work; Xenopus; Xenopus laevis; axon growth; axon guidance; axonal guidance; base; career; career development; cell motility; combinatorial; design; experience; extracellular; gain of function; genetic analysis; genetic manipulation; in vivo; innovation; loss of function; medical schools; neural circuit; neurogenesis; neuronal growth; neuropsychiatry; pre-doctoral; programs; public health relevance; relating to nervous system; research and development; research facility; research study; skills; success; tool; treatment strategy

The long-term goal of Dr. Laura Anne Lowery is to obtain a tenure-track faculty position at a research university and develop a comprehensive, multi-faceted research program that investigates the logic by which guidance information is integrated at the level of cytoskeletal dynamics during axon pathfinding. To this end, she has constructed an extensive career development and research training plan which will facilitate her success and complement her previous training experiences. She received her BS and MS in biology from UCSD, where she worked with Dr. William Schafer on the neural circuitry controlling C. elegans behavior. This work resulted in two papers (including first-author in Journal of Neurobiology). She received her PhD in Biology at MIT under the mentorship of Dr. Hazel Sive. Supported by a pre-doctoral NRSA, she made significant progress defining the genes essential for early brain morphogenesis, including the identification of several genes required for normal neurogenesis and axon pathway formation. This work resulted in five first- author publications in journals such as Development. In July 2008, Dr. Lowery joined the Van Vactor lab in the Department of Cell Biology at Harvard Medical School, where she began a project to identify new interactors of an intriguing cytoskeletal regulator that functions downstream of axon guidance cues, called CLASP. This work, supported by a post-doctoral NRSA, has thus far resulted in 2 first-author publications (in Genetics and Nature Reviews). Dr. Lowery's immediate goal is to gain new expertise in quantitative cytoskeletal imaging and analysis using Xenopus growth cones, in order to investigate the roles of specific microtubule regulators during axon guidance. While in the mentored K99 phase, Dr. Lowery will continue to benefit from the mentorship of Dr. Van Vactor, a leader in the field of genetic analysis of axonal growth and guidance. Additionally, Dr. Lowery will receive new training and support from co-mentor Dr. Gaudenz Danuser, one of the world's leaders in quantitative cytoskeletal analysis. Both Drs. Van Vactor and Danuser have excellent mentoring records and are committed to fostering Dr. Lowery's training and independence. This environment is an ideal setting for her transition to independence, as Harvard Medical School is one of the strongest biomedical research facilities in the country and is perfectly suited to facilitate the goals in this proposal. Her development will be enhanced by additional microscopy and computation courses, as well as support from an advisory committee of expert investigators of axon guidance and the cytoskeleton. The new skills, techniques, and experimental data she acquires during the K99 phase (Aims 1, 2) are essential to the research planned for the independent R00 phase (Aim 3). The research objective in this application is to determine how a specific group of microtubule 'plus-end tracking proteins' (+TIPs) localize, interact, and function, within the growth cone downstream of guidance cue signaling. Initial work has identified +TIP XMAP215 and its co-factor Maskin as potent antagonists of the +TIP and Abl signaling substrate, CLASP. Furthermore, XMAP215 and Maskin are required for accurate axon guidance decisions in vivo, and XMAP215 antagonizes Abl's in vivo axon guidance function. These preliminary findings, combined with knowledge from non-neuronal studies of +TIP function, have led to the working model that, within the growth cone, XMAP215 and Maskin interact with microtubules (MTs) in a functionally-distinct manner compared to CLASP, and that Abl signaling leads to differences in the ability of these +TIPs to interact with each other and with microtubules, thereby driving changes in cytoskeletal dynamics and growth cone directionality downstream of guidance cues. This will be tested using a combination of quantitative imaging, genetic manipulations, and biochemical approaches, to pursue three specific aims. Aim 1) How do +TIPs behave and co-localize with each other and with microtubules inside the growth cone? +TIP localization and MT dynamic instability parameters will be quantified using computational analysis, following acquisition of high-resolution live imaging data of +TIPs and MTs within cultured Xenopus growth cones. Aim 2) How does +TIP function influence MT dynamics and growth cone motility? This aim will use loss-of-function and gain-of-function genetic strategies in Xenopus combined with the imaging platform established in Aim 1 to identify the functional roles of XMAP215 and Maskin, compared to CLASP, within the growth cone. Aim 3) How is +TIP function within the growth cone regulated by upstream guidance signaling? In part 3A, biochemical experiments using Xenopus embryonic lysates will be performed to assess the regulation of +TIP binding events in vitro and to determine the structural domains that modulate those interactions. In part 3B, high-resolution live imaging will allow visualization of +TIP/MT interactions as the growth cone encounters guidance cues in culture, as well as after direction manipulation of Abl signaling. This approach is innovative because it will, for the first time, combine state-of-the-art imaging and analysis tools to pioneer the elucidation of quantitative global MT and +TIP behavior within cultured growth cones during decision-making events. The proposed research is significant because it is an important step in a continuum of research that will illuminate how the growth cone cytoskeleton is coordinated during axon guidance, the knowledge of which may eventually be applied to understanding the basis of neurodevelopmental and mental health disorders.

Laura Anne Lowery 博士的长期目标是在一家研究机构获得终身教职 大学并制定一个全面的、多方面的研究计划，调查其逻辑 在轴突寻路过程中，引导信息在细胞骨架动力学水平上进行整合。为此， 她制定了广泛的职业发展和研究培训计划，这将有助于她 成功并补充了她之前的培训经验。她获得了生物学学士和硕士学位 加州大学圣地亚哥分校，她与 William Schafer 博士一起研究控制线虫行为的神经回路。这 工作成果包括两篇论文（包括《Journal of Neurobiology》的第一作者）。她获得了博士学位 在 Hazel Sive 博士的指导下在麻省理工学院学习生物学。在博士前 NRSA 的支持下，她做了 定义早期大脑形态发生所必需的基因的重大进展，包括识别 正常神经发生和轴突通路形成所需的几个基因。这项工作获得了五个第一—— 在《发展》等期刊上发表论文。 2008 年 7 月，Lowery 博士加入 Van Vactor 实验室 哈佛医学院细胞生物学系，她在那里启动了一个项目来识别新的相互作用者 一种有趣的细胞骨架调节器，在轴突引导信号的下游发挥作用，称为 CLASP。这 这项工作得到了 NRSA 博士后的支持，迄今为止已发表了 2 篇第一作者出版物（《遗传学和遗传学》） 自然评论）。 Lowery 博士的近期目标是获得定量细胞骨架成像方面的新专业知识 并使用爪蟾生长锥进行分析，以研究特定微管调节因子的作用 在轴突引导期间。在 K99 指导阶段，Lowery 博士将继续受益于 Van Vactor 博士是轴突生长和指导遗传分析领域的领导者。 此外，Lowery 博士还将接受联合导师 Gaudenz Danuser 博士的新培训和支持，Gaudenz Danuser 博士是 定量细胞骨架分析领域的世界领先者。两位博士。范·瓦克托 (Van Vactor) 和丹努瑟 (Danuser) 表现出色 指导记录并致力于培养 Lowery 博士的培训和独立性。这个环境 哈佛医学院是她过渡到独立的理想环境，因为哈佛医学院是最强大的学院之一 该国的生物医学研究设施非常适合促进本提案中的目标。她 额外的显微镜和计算课程以及来自 轴突引导和细胞骨架专家调查员咨询委员会。新的技能、技术、 她在 K99 阶段（目标 1、2）获得的实验数据对于计划的研究至关重要 独立的 R00 阶段（目标 3）。本申请的研究目标是确定特定的 一组微管“正端追踪蛋白”(+TIP) 在生长锥内定位、相互作用并发挥作用 引导提示信号的下游。初步工作已确定 +TIP XMAP215 及其辅因子 Maskin 为 +TIP 和 Abl 信号传导底物 CLASP 的有效拮抗剂。此外，XMAP215 和 Maskin 是 体内准确的轴突引导决策所需，并且 XMAP215 拮抗 Abl 的体内轴突引导 功能。这些初步发现与 +TIP 功能的非神经元研究知识相结合， 得出了这样的工作模型：在生长锥内，XMAP215 和 Maskin 与微管相互作用 (MT) 与 CLASP 相比，以功能不同的方式，并且 Abl 信号传导导致 这些 +TIP 之间以及与微管相互作用的能力，从而驱动细胞骨架的变化 引导线索下游的动力学和生长锥方向性。这将使用一个测试 结合定量成像、基因操作和生化方法，追求三种 具体目标。目标 1) +TIP 之间以及与微管内的微管如何表现和共定位 生长锥？ +TIP 定位和 MT 动态不稳定性参数将通过计算进行量化 采集培养爪蟾内 +TIP 和 MT 的高分辨率实时成像数据后进行分析 生长锥。目标 2) +TIP 功能如何影响 MT 动力学和生长锥运动？这一目标将 结合成像平台在非洲爪蟾中使用功能丧失和功能获得遗传策略 目标 1 中确定了与 CLASP 相比，XMAP215 和 Maskin 的功能作用，在 生长锥。目标 3) 生长锥内的 +TIP 功能如何受到上游引导信号的调节？ 在第 3A 部分中，将使用非洲爪蟾胚胎裂解物进行生化实验来评估 体外调节 +TIP 结合事件并确定调节这些事件的结构域 互动。在第 3B 部分中，高分辨率实时成像将允许 +TIP/MT 交互的可视化 生长锥在培养中以及在 Abl 信号传导的方向操纵后遇到引导线索。这 该方法具有创新性，因为它将首次将最先进的成像和分析工具结合起来 率先阐明了培养生长锥内的定量全局 MT 和 +TIP 行为 决策事件。拟议的研究意义重大，因为它是连续过程中的重要一步 研究将阐明生长锥细胞骨架在轴突引导过程中如何协调， 这些知识最终可以应用于理解神经发育和心理的基础 健康障碍。