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: 8795223
• 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/8795223
• 关键词: Advisory Committees; Affect; Autistic Disorder; Automobile Driving; Axon; Behavior; Binding; Biochemical; Biological Assay; Biology; Biomedical Research; Brain; Caenorhabditis elegans; Cellular biology; 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; imaging platform; in vivo; innovation; loss of function; medical schools; neural circuit; neurogenesis; neuronal growth; neuropsychiatry; pre-doctoral; programs; public health relevance; quantitative imaging; 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.

劳拉·安妮·洛里（Laura Anne Lowery）博士的长期目标是在研究中获得终身教师职位 大学并制定一项全面的，多面的研究计划，该计划调查了逻辑 引导信息集成在轴突探测过程中的细胞骨架动力学水平上。为此， 她建立了广泛的职业发展和研究培训计划，这将促进她 成功并补充她以前的培训经验。她从 UCSD，她与William Schafer博士一起在控制秀丽隐杆线虫行为的神经电路上工作。这 作品产生了两篇论文（包括神经生物学杂志的第一任作者）。她获得了博士学位 在Hazel Sive博士的指导下，MIT的生物学。在博士前NRSA的支持下，她做了 定义早期脑形态发生必不可少的基因的重大进展，包括鉴定 正常神经发生和轴突途径形成所需的几种基因。这项工作导致了五个 诸如开发等期刊的作者出版物。 2008年7月，Lowery博士加入了Van Vactor实验室 哈佛医学院的细胞生物学系，在那里她开始了一个项目，以确定新的互动者 一个有趣的细胞骨架调节剂，在轴突引导线索下游发挥作用，称为clasp。这 在博士后NRSA的支持下的工作，到目前为止，已有2份第一名第一专有出版物（遗传学和遗传学和 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剂的作用 在轴突指导期间。在指导的K99阶段，Lowery博士将继续从中受益 Van Vactor博士的指导是轴突生长和指导遗传分析领域的领导者。 此外，Lowery博士将获得Gaudenz Danuser博士的新培训和支持， 定量细胞骨架分析的世界领导者。两个博士。 Van Vactor和Danuser的出色 指导记录，并致力于培养Lowery博士的培训和独立性。这个环境 是她向独立过渡的理想环境，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该提案中的目标。她 其他显微镜和计算课程将增强开发，以及 Axon Guidance and the Cytoskeleton的专家调查员咨询委员会。新技能，技术， 她在K99阶段获得的实验数据（目标1，2）对于计划的研究至关重要 独立R00阶段（AIM 3）。本应用程序中的研究目标是确定特定的 在生长锥中，微管“加末端跟踪蛋白”（+TIPS）本地化，相互作用和功能 指南信号的下游。最初的工作已确定 +TIP XMAP215及其coactor Maskin是 +尖端的有效拮抗剂和ABL信号基板扣子。此外，XMAP215和Maskin是 在体内准确的轴突指导决策所必需的，XMAP215拮抗ABL的体内轴突指导 功能。这些初步发现，加上来自 +尖端功能的非神经元研究的知识， 导致了一个工作模型，该模型在生长锥中XMAP215和Maskin与微管相互作用 （MT）与扣子相比具有功能上的方式，并且ABL信号传导导致差异 这些 +尖端彼此相互作用并与微管相互作用的能力，从而驱动细胞骨架的变化 指导线索下游的动态和生长锥方向。这将使用 定量成像，遗传操纵和生化方法的结合，追求三个 具体目标。目标1） +尖端如何表现并彼此共定位 生长锥？ +TIP定位和MT动态不稳定性参数将使用计算进行量化 分析，在培养的Xenopus中获得 +TIPS和MTS的高分辨率实时成像数据后 生长锥。目标2） +尖端功能如何影响MT动力学和生长锥运动？这个目标 在Xenopus中使用功能丧失和功能获得的遗传策略与成像平台相结合 与扣子相比 生长锥。目标3）在上游指导信号调节的生长锥中 +尖端功能如何？ 在第3A部分中，将进行使用爪蟾胚胎裂解物的生化实验，以评估 在体外调节 +尖端结合事件，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可视化 +tip/mt相互作用作为 生长锥遇到文化中的引导提示以及ABL信号的后方操纵。这 方法具有创新性，因为它将首次将最新成像和分析工具结合起来 先驱在培养的生长锥中阐明定量全球MT和 +尖端行为 决策事件。拟议的研究很重要，因为它是连续性的重要一步 将阐明生长锥细胞骨架如何协调期间如何协调的研究， 知识最终可以应用于理解神经发育和精神的基础 健康障碍。