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

描述（由申请人提供）：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，多方面的研究项目，研究轴突寻路过程中在细胞骨架动力学水平上整合指导信息的逻辑。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博士一起研究控制秀丽隐杆线虫行为的神经回路。这项工作产生了两篇论文（包括第一作者在Journal of Neurobiology）。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，包括确定正常神经发生和轴突通路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，Lowery博士加入了哈佛医学院细胞生物系的Van Vactor实验室，在那里她开始了一个项目，寻找一种有趣的细胞骨架调节剂的新相互作用物，这种调节剂在轴突引导信号的下游起作用，称为CLASP。这项工作得到了博士后NRSA的支持，迄今已在《遗传学》和《自然评论》上发表了两篇第一作者论文。Lowery博士的近期目标是利用爪蟾生长锥获得定量细胞骨架成像和分析的新专业知识，以研究特定微管调节剂在轴突引导过程中的作用。在受指导的K99阶段，Lowery博士将继续受益于Van Vactor博士的指导，Van Vactor博士是轴突生长遗传分析和指导领域的领导者。此外，Lowery博士将接受联合导师Gaudenz Danuser博士的新培训和支持，他是定量细胞骨架分析领域的世界领导者之一。Drs。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。这个环境是她向独立过渡的理想环境，因为哈佛医学院是全国最强大的生物医学研究设施之一，非常适合促进这项提案的目标。她的发展将通过额外的显微镜和计算课程，以及轴突指导和细胞骨架专家研究人员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、技术和实验数据对独立的R00阶段（目标3）的研究计划至关重要。本应用的研究目的是确定一组特定的微管“+端跟踪蛋白”（+TIPs）如何在引导线索信号下游的生长锥内定位、相互作用和起作用。最初的研究已经确定+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在体内需要精确的轴突引导决策，而XMAP215在体内拮抗Abl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，得出了一个工作模型，即在生长锥内，XMAP215和Maskin以与CLASP不同的功能方式与微管（MTs）相互作用，并且Abl信号导致这些+TIPs相互作用能力的差异以及与微管的相互作用，从而驱动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，遗传操作和生化方法的组合进行测试，以追求三个具体目标。目标1)+TIPs如何相互作用并与生长锥内的微管共定位？在获取培养爪蟾生长锥内+TIPs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？本目标将使用爪蟾的功能丧失和功能获得遗传策略，结合aim 1中建立的成像平台，确定XMAP215和Maskin在生长锥内的功能作用，与CLASP相比。目标3)生长锥内的+TIP功能如何受到上游引导信号的调节？在第3A部分，将使用非洲爪蟾胚胎裂解液进行生化实验，以评估+TIP结合事件在体外的调节作用，并确定调节这些相互作用的结构域。在第3B部分中，高分辨率实时成像将允许可视化+TIP/MT相互作用，当生长锥在培养中遇到引导线索时，以及在Abl信号的方向操纵之后。这种方法是创新的，因为它将首次结合尖端成像和分析工具，率先阐明决策过程中培养生长锥内定量的全球MT和+TIP行为。拟议的研究是重要的，因为它是一个连续研究的重要步骤，将阐明如何

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.