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: 8215540
• 负责人: Laura Anne LOWERY
• 金额: $ 9.1万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-02-06 至 2014-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8215540
• 关键词: Advisory Committees; Affect; 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; Doctor of Philosophy; Embryo; Environment; Event; Faculty; Fostering; Frequencies; Genes; Genetic; Goals; Growth; Growth Cones; Image; Image Analysis; Imagery; In Vitro; Journals; Knowledge; 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; Protein Family; Protein Tyrosine Kinase; Proteins; Publications; Records; Regulation; Research; Research Personnel; Research Training; Resolution; Retinal Cone; Role; 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; experience; extracellular; gain of function; genetic analysis; genetic manipulation; in vivo; innovation; loss of function; medical schools; neural circuit; neurogenesis; neuronal growth; pre-doctoral; programs; relating to nervous system; research and development; research facility; research study; skills; success; tool

DESCRIPTION (provided by applicant): 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-ar 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. PUBLIC HEALTH RELEVANCE: Project Narrative/Public Health Relevance Statement Proper neural connections are essential for normal nervous system function. Abnormalities in neural connectivity are associated with a multitude of neurodevelopmental and mental health disorders, including autism and schizophrenia. Determining the mechanism by which growth cones are guided during axon pathfinding may eventually lead to understanding the basis of neuropsychiatric disorders and may contribute to designing prevention and/or treatment strategies in the future.

描述（由申请人提供）：Laura Anne Lowery博士的长期目标是在研究大学获得终身教师职位，并制定一项全面的，多方面的研究计划，该计划调查了在Axon Pathfinding期间通过该逻辑集成在该逻辑上，该逻辑通过该逻辑进行了逻辑。为此，她构建了广泛的职业发展和研究培训计划，这将促进她的成功并补充她以前的培训经验。她从UCSD获得了BS和MS的生物学学士学位，并在那里与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。这项工作产生了两篇论文（包括神经生物学杂志的第一任作者）。在Hazel Sive博士的指导下，她在MIT获得了生物学博士学位。在博物前NRSA的支持下，她取得了重大进展，以定义早期脑形态发生必不可少的基因，包括鉴定出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在开发等期刊上的第一作者出版物。 2008年7月，Lowery博士加入了哈佛医学院细胞生物学系的Van Vactor实验室，在那里她开始了一个项目，以确定一个有趣的细胞骨架调节器的新互动，该互动剂在Axon Guidance Cues下游起作用，称为CLASP。迄今为止，这项工作得到了博士后NRSA的支持。 Lowery博士的近期目标是使用异爪蟾生长锥获得定量细胞骨架成像和分析的新专业知识，以研究在轴突指导过程中特定微管调节剂的作用。在指导的K99阶段，Lowery博士将继续受益于Van Vactor博士的指导，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博士将获得全球定量细胞骨架分析领导者之一Gaudenz Danuser博士的新培训和支持。两个博士。 Van Vactor和Danuser拥有出色的指导记录，并致力于培养Lowery博士的培训和独立性。这种环境是她过渡到独立性的理想环境，因为哈佛医学院是该国最强的生物医学研究机构之一，非常适合促进该提案中的目标，并通过其他显微镜和计算课程以及Axon Guidance and Cytoskeleton的专家咨询委员会的支持来增强她的发展。她在K99阶段获得的新技能，技术和实验数据（AIMS 1，2）对于计划的独立R00阶段的研究至关重要（AIM 3）。本应用程序中的研究目标是确定特定的微管“加上末端跟踪蛋白”（+TIPS）如何本地化，相互作用和功能在指导提示信号下游的生长锥内。最初的工作已将 +尖端XMAP215及其coactor Maskin确定为 +尖端和ABL信号底物的有效拮抗剂。此外，XMAP215和Maskin在体内需要准确的轴突指导决策，而XMAP215拮抗了ABL的体内轴突指导函数。这些初步发现，再加上来自非尖端功能的非神经元研究的知识，导致了工作模型，该模型在生长锥中XMAP215和Maskin与clasp相比，以功能固定的方式与微管（MTS）相互作用，并且ABL信号与这些 +tip的能力相互作用，并与其他端口互动，并与其他人相互作用。以及指导线索下游的生长锥方向性。这将通过定量成像，遗传操作和生化方法的组合来实现这一测试，以追求三个特定目标。目标1） +尖端如何表现并与生长锥内部的微管共定位？ +尖端定位和MT动态不稳定性参数将在获取培养的Xenopus生长锥内 +TIPS和MT的高分辨率实时成像数据后，使用计算分析进行量化。目标2） +尖端功能如何影响MT动力学和生长锥运动？与AIM 1中建立的成像平台相比，该目标将使用Xenopus中的功能丧失和功能获得遗传策略，以识别XMAP215和Maskin的功能作用，与cLASP相比，在生长锥中与cLASP相比。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，将进行使用异武胚裂解物的生化实验，以评估体外的 +尖端结合事件的调节，并确定调节这些相互作用的结构域。在第3B部分中，高分辨率的实时成像将允许可视化 +尖端/MT相互作用，因为生长锥遇到培养中的引导线索以及ABL信号传导的后方向操纵。这种方法具有创新性，因为它将首次将最新成像和分析工具结合起来，以开拓决策事件期间培养的生长锥体内定量的全球MT和 +尖端行为。拟议的研究很重要，因为它是连续研究的重要一步，它将阐明如何阐明如何 在轴突指导期间，生长锥细胞骨架是协调的，这些知识最终可用于理解神经发育和心理健康障碍的基础。 公共卫生相关性：项目叙事/公共卫生相关性声明适当的神经联系对于正常神经系统功能至关重要。神经连通性的异常与包括自闭症和精神分裂症在内的多种神经发育和心理健康障碍有关。确定在轴突探测过程中指导生长锥的机制最终可能导致理解神经精神疾病的基础，并可能有助于设计预防和/或将来的治疗策略。