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: 8420338
• 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/8420338
• 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.

Laura Anne Lowery博士的长期目标是在一所研究型大学获得终身教职，并开发一个全面的，多方面的研究计划，该计划调查指导信息在轴突寻路过程中在细胞骨架动力学水平上整合的逻辑。为此，她制定了一个广泛的职业发展和研究培训计划，这将有助于她的成功，并补充她以前的培训经验。她在加州大学圣地亚哥分校获得生物学学士和硕士学位，在那里她与William Schafer博士一起研究控制C。优雅的行为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）。她在麻省理工学院获得生物学博士学位，师从Hazel Sive博士。在博士前NRSA的支持下，她在定义早期脑形态发生所必需的基因方面取得了重大进展，包括鉴定了正常神经发生和轴突通路形成所需的几个基因。这项工作的成果是在《发展》等期刊上发表了五篇第一作者论文。2008年7月，Lowery博士加入了哈佛医学院细胞生物学系的货车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相互作用，该调节器在轴突引导线索下游发挥作用，称为CLASP。这项工作得到了博士后NRSA的支持，到目前为止，已经有2篇第一作者出版物（遗传学和自然评论）。Lowery博士的近期目标是利用非洲爪蟾生长锥获得定量细胞骨架成像和分析的新专业知识，以研究轴突引导过程中特定微管调节剂的作用。在指导K99阶段，Lowery博士将继续受益于货车Vactor博士的指导，他是轴突生长和指导遗传分析领域的领导者。此外，Lowery博士将接受来自共同导师Gaudenz Danuser博士的新培训和支持，他是定量细胞骨架分析的世界领导者之一。货车Vactor博士和Danuser博士都有出色的指导记录，并致力于培养Lowery博士的培训和独立性。这种环境是她向独立过渡的理想环境，因为哈佛医学院是美国最强大的生物医学研究机构之一，非常适合实现本提案中的目标。她的发展将通过额外的显微镜和计算课程以及轴突引导和细胞骨架专家研究人员咨询委员会的支持得到加强。她在K99阶段（目标1，2）获得的新技能，技术和实验数据对于独立R 00阶段（目标3）计划的研究至关重要。本申请的研究目标是确定一组特定的微管“+末端跟踪蛋白”（+TIPs）如何在生长锥下游的引导信号传导中定位、相互作用和发挥作用。最初的工作已经确定+TIP XMAP 215及其辅因子Maskin是+TIP和Abl信号传导底物CLASP的有效拮抗剂。此外，XMAP 215和Maskin是体内精确轴突引导决定所必需的，并且XMAP 215拮抗Abl的体内轴突引导功能。这些初步发现，结合来自+TIP功能的非神经元研究的知识，已经导致了工作模型，即在生长锥内，与CLASP相比，XMAP 215和Maskin以功能上不同的方式与微管（MT）相互作用，并且Abl信号传导导致这些+TIP彼此相互作用以及与微管相互作用的能力的差异，从而驱动细胞骨架动力学和导向线索下游的生长锥方向性的变化。这将使用定量成像，遗传操作和生物化学方法的组合进行测试，以追求三个特定的目标。目的1）+TIPs在生长锥内的行为和相互之间以及与微管的共定位如何？在采集培养的非洲爪蟾生长锥内+TIP和MT的高分辨率实时成像数据后，将使用计算分析对+TIP定位和MT动态不稳定性参数进行量化。目的2）+TIP功能如何影响MT动力学和生长锥运动？该目标将使用爪蟾中的功能丧失和功能获得遗传策略，结合目标1中建立的成像平台，以确定与CLASP相比，XMAP 215和Maskin在生长锥内的功能作用。目的3）+TIP在生长锥内的功能是如何被上游引导信号调控的？在第3A部分中，将使用非洲爪蟾胚胎裂解物进行生化实验，以评估+TIP结合事件的体外调节，并确定调节这些相互作用的结构域。在部分3B中，高分辨率实时成像将允许随着生长锥在培养中遇到引导线索以及在Abl信号传导的方向操纵之后可视化+TIP/MT相互作用。这种方法是创新的，因为它将首次将联合收割机最先进的成像和分析工具结合起来，开创性地阐明了决策事件期间培养的生长锥内的定量全局MT和+TIP行为。拟议的研究是重要的，因为它是一个连续的研究，将阐明如何 生长锥细胞骨架在轴突引导过程中协调一致，这方面的知识最终可能被应用于理解神经发育和精神健康障碍的基础。