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.

劳拉·安妮·洛厄里博士的长期目标是在一项研究中获得终身教职 大学，并开发一个全面的、多方面的研究计划，调查 在轴突寻路过程中，引导信息在细胞骨架动力学水平上被整合。为此， 她构建了广泛的职业发展和研究培训计划，这将有助于她 成功，并补充了她以前的培训经验。她从哈佛大学获得生物学学士和硕士学位。 在加州大学洛杉矶分校，她与威廉·谢弗博士合作研究控制线虫行为的神经电路。这 这项工作产生了两篇论文(包括《神经生物学杂志》的第一作者)。她于年获得博士学位。 在黑兹尔·西夫博士的指导下，麻省理工学院的生物学。在一位博士后NRSA的支持下，她做出了 确定早期脑形态发生所必需基因的重大进展，包括鉴定 正常神经发生和轴突形成所需的几个基因。这项工作产生了五个第一- 在《发展》等期刊上发表论文。2008年7月，洛厄里博士加入范·维泰尔实验室 哈佛医学院细胞生物学系，在那里她开始了一个项目，以确定新的相互作用因素 一种耐人寻味的细胞骨架调节器，其功能位于轴突引导信号下游，称为CLASP。这 在一名博士后NRSA的支持下，到目前为止已经发表了两篇第一作者出版物(《遗传学》和《 《自然评论》)。洛厄里博士的近期目标是在定量细胞骨架成像方面获得新的专业知识 并使用非洲爪哇生长锥体进行分析，以研究特定微管调节因子的作用 在轴突引导过程中。在K99指导阶段，Lowery博士将继续受益于 指导凡·瓦泰尔博士，他是轴突生长和指导基因分析领域的领先者。 此外，洛厄里博士还将接受合作导师高登兹·丹尼瑟博士的新培训和支持，丹尼瑟博士是 在定量细胞骨架分析方面处于世界领先地位。凡·维泰尔博士和丹尼瑟博士都有出色的 并致力于培养洛厄里博士的训练能力和独立性。这个环境 是她过渡到独立的理想环境，因为哈佛医学院是最强大的之一 中国拥有生物医学研究设施，非常适合促进这项提案中的目标。她 开发将通过增加显微镜和计算课程以及来自 轴突指导和细胞骨架专家调查咨询委员会。新的技能，技巧， 她在K99阶段(目标1、2)获得的实验数据对于计划的研究是必不可少的 独立的R00阶段(目标3)。本应用程序的研究目标是确定特定的 一组微管‘+末端跟踪蛋白’(+TIPS)在生长锥体内定位、相互作用并发挥作用 在引导信号的下游。初步工作已确定+TIP XMAP215及其辅助因子掩蔽为 +TIP和Abl信号底物的有效拮抗剂CLASP。此外，XMAP215和Maskin是 在活体内做出准确的轴突引导决定所需的，并且XMAP215拮抗Abl在体内的轴突引导 功能。这些初步发现，结合对+TIP功能的非神经元研究的知识， 导致了在生长锥内，XMAP215和Maskin与微管相互作用的工作模型 (MTS)与CLAP相比，在功能上是不同的，ABL信号导致了 这些+尖端相互作用并与微管相互作用的能力，从而推动细胞骨架的变化 指导线索下游的动态和生长锥体方向性。这将使用 结合定量成像、基因操作和生化方法，追求三个 明确的目标。目标1)+尖端如何相互作用和协同定位，以及如何与微管共同定位 生长锥+针尖局部化和MT动态不稳定性参数将通过计算 在获得非洲爪哇养殖中+TIPS和MTS的高分辨率实时成像数据后进行分析 生长锥体。目的2)+TIP功能对MT动力学和生长锥运动有何影响？这一目标将 结合成像平台在非洲爪哇中使用功能丧失和功能获得遗传策略 在目标1中建立，以确定XMAP215和Maskin相对于CLAP在 生长锥体。目的3)+TIP在生长锥内的功能如何受上游引导信号的调节？ 在第3A部分，将使用非洲爪哇胚胎裂解物进行生化实验，以评估 体外调节+TIP结合事件并确定调节这些结合事件的结构域 互动。在第3B部分中，高分辨率实时成像将允许可视化+TIP/MT交互作用 生长锥在培养中遇到指导线索，以及在Abl信号的方向操纵之后。这 方法是创新的，因为它将首次将最先进的成像和分析工具结合在一起，以 率先阐明了培养的生长锥体内MT和+TIP的定量行为 决策事件。这项拟议的研究具有重要意义，因为它是 这项研究将阐明生长锥体细胞骨架在轴突引导过程中是如何协调的 其中的知识最终可能被用于理解神经发育和心理的基础 健康障碍。