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

描述(由申请者提供)：Laura Anne Lowery博士的长期目标是在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，调查轴突寻径过程中指导信息在细胞骨架动力学水平上的整合逻辑。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，并补充她之前的培训经验。她在加州大学圣迭戈分校获得生物学学士和硕士学位，在那里她与威廉·谢弗博士合作研究控制线虫行为的神经电路。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)。在黑兹尔·西维博士的指导下，她在麻省理工学院获得了生物学博士学位。在一名博士前NRSA的支持下，她在确定早期大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路径形成所需的几个基因。这项工作在《发展》等期刊上发表了五篇第一作者论文。2008年7月，洛厄里加入了哈佛医学院细胞生物学系的范·瓦泰尔实验室，在那里她开始了一个项目，目的是寻找一种有趣的细胞骨架调节因子的新相互作用因子，这种调节因子的功能是轴突引导信号CLASP的下游。这项工作得到了一位博士后NRSA的支持，到目前为止已经发表了两篇第一作者论文(《遗传学》和《自然评论》)。洛厄里博士的近期目标是在利用非洲爪哇生长锥体进行定量细胞骨架成像和分析方面获得新的专业知识，以便研究特定微管调节剂在轴突引导过程中的作用。在K99指导阶段，洛厄里博士将继续受益于凡·瓦泰尔博士的指导，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。丹尼瑟博士是定量细胞骨架分析领域的世界领先者之一。范·维泰尔博士和丹尼瑟博士都有出色的指导记录，并致力于培养洛厄里博士的训练和独立性。这种环境是她过渡到独立的理想环境，因为哈佛医学院是美国最强大的生物医学研究机构之一，非常适合促进这项提案中的目标她的发展将通过额外的显微镜和计算课程，以及来自轴突指导和细胞骨架专家调查咨询委员会的支持而得到加强。她在K99阶段(目标1、2)获得的新技能、技术和实验数据对于为独立R00阶段(目标3)计划的研究至关重要。这项应用的研究目标是确定一组特定的微管‘+末端跟踪蛋白’(+TIPS)如何在引导信号下游的生长锥内定位、相互作用和发挥功能。初步工作已确定+TIP XMAP215及其辅助因子Maskin是+TIP和Abl信号底物CLASP的有效拮抗剂。此外，XMAP215和Maskin是体内准确的轴突引导决策所必需的，而XMAP215拮抗Abl的体内轴突引导功能。结合这些初步发现，结合对+TIP功能的非神经元研究，建立了这样的工作模型：在生长锥内，XMAP215和Maskin以一种与CLAP相比在功能上不同的方式与微管(MTS)相互作用，并且Abl信号导致这些+TIP相互作用和与微管相互作用的能力不同，从而推动指导信号下游细胞骨架动力学和生长锥方向性的变化。这将使用定量成像、基因操作和生化方法的组合进行测试，以追求三个特定的目标。目的1)+TIPS如何在生长锥体内与微管相互作用和协同定位？+TIPS的定位和MT动态不稳定性参数将通过计算分析，在获得培养的非洲爪哇生长锥内+TIPS和MTS的高分辨率实时成像数据后定量。目的2)+TIP功能对MT动力学和生长锥运动有何影响？这个目标将使用非洲爪哇的功能丧失和功能获得遗传策略，结合目标1中建立的成像平台来确定XMAP215和Maskin相对于CLAP在生长锥体中的功能作用。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3A部分，将使用非洲爪哇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3B部分中，当生长锥在培养中遇到指导线索时，高分辨率的实时成像将允许可视化+TIP/MT的相互作用，以及ABL信号的方向操纵之后。这种方法是创新的，因为它将首次结合AR状态成像和分析工具，率先阐明在决策事件期间培养的生长锥体内定量的全球MT和+TIP行为。这项拟议的研究意义重大，因为它是一系列研究中的重要一步，将阐明 生长锥细胞骨架在轴突引导过程中是协调的，其知识最终可能被应用于理解神经发育和心理健康障碍的基础。

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.