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

劳拉·安妮·洛厄里博士的长期目标是在一个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，调查的逻辑， 在轴突寻路期间，在细胞骨架动力学水平上整合引导信息。为此，委员会建议， 她已经建立了一个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。她获得了生物学学士和硕士学位， 在那里，她与威廉·谢弗博士一起研究控制C。优雅的行为这 工作产生了两篇论文（包括第一作者在神经生物学杂志）。她获得了博士学位， 在麻省理工学院的生物学博士的指导下，黑兹尔西夫。在博士前NRSA的支持下，她 在确定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 正常神经发生和轴突通路形成所需的几种基因。这项工作取得了五个第一- 在《发展》等期刊上发表文章。2008年7月，Lowery博士加入了货车Vactor实验室， 在哈佛医学院的细胞生物学系，她开始了一个项目，以确定新的相互作用， 一个有趣的细胞骨架调节器，在轴突引导线索的下游发挥作用，称为CLASP。这 工作，由博士后NRSA的支持，迄今已导致2第一作者出版物（遗传学和 Nature Reviews）. Lowery博士的近期目标是获得定量细胞骨架成像的新专业知识 并使用非洲爪蟾生长锥进行分析，以研究特定微管调节剂的作用 在轴突的引导下。在K99辅导阶段，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博士将接受新的培训，并得到共同导师Gaudenz Danuser博士的支持， 细胞骨架定量分析领域的世界领先者。货车·瓦克特博士和丹瑟博士都有很好的 指导记录，并致力于促进博士Lowery的培训和独立性。这种环境 是她向独立过渡的理想环境，因为哈佛医学院是最强大的医学院之一。 这是该国最大的生物医学研究设施，完全适合促进本提案中的目标。她 发展将得到加强，额外的显微镜和计算课程，以及支持， 轴突导向和细胞骨架专家研究咨询委员会。新的技能，技术， 她在K99阶段获得的实验数据（目标1，2）对计划进行的研究至关重要。 独立R 00阶段（目标3）。本申请的研究目标是确定特定的 一组微管“+末端跟踪蛋白”（+TIPs）在生长锥内定位、相互作用和发挥功能 引导提示信号的下游。最初的工作已经确定+TIP XMAP 215及其辅助因子Maskin为 +TIP和Abl信号传导底物CLASP的有效拮抗剂。此外，XMAP 215和Maskin 体内精确的轴突导向决定所需的，并且XMAP 215拮抗Abl的体内轴突导向 功能这些初步发现，结合非神经元研究+TIP功能的知识， 已经导致了工作模型，在生长锥内，XMAP 215和Maskin与微管相互作用 (MTs)与CLASP相比，Abl信号传导导致了在CLASP中的差异。 这些+TIPs相互作用以及与微管相互作用的能力，从而驱动细胞骨架的变化， 动力学和生长锥方向性下游的指导线索。这将使用 定量成像，遗传操作和生物化学方法的组合，以追求三个 具体目标。目的1）+TIPs是如何行为的，如何相互共定位，以及如何与微管共定位。 生长锥+TIP定位和MT动态不稳定参数将使用计算 分析，采集培养的非洲爪蟾内+TIP和MT的高分辨率实时成像数据后 生长锥目的2）+TIP功能如何影响MT动力学和生长锥运动？这一目标将 在非洲爪蟾中使用功能丧失和功能获得遗传策略结合成像平台 目的1中建立的，以确定XMAP 215和Maskin的功能角色，与CLASP相比， 生长锥目的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，将使用非洲爪蟾胚胎裂解物进行生化实验，以评估 在体外调节+TIP结合事件，并确定调节这些结合事件的结构域。 交互.在第3B部分中，高分辨率实时成像将允许+TIP/MT相互作用的可视化， 生长锥在培养中以及在Abl信号传导的方向操纵之后遇到引导线索。这 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， 开创了在培养的生长锥中定量的全局MT和+TIP行为的阐明， 决策事件。拟议的研究是重要的，因为它是一个连续的重要一步， 这项研究将阐明生长锥细胞骨架在轴突引导过程中是如何协调的， 这方面的知识最终可能被应用于理解神经发育和心理的基础。 健康失调

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.