Regulation of Neuronal Growth Cone Guidance

神经元生长锥引导的调节

• 批准号: 7216183
• 负责人: DANIEL Marcel SUTER
• 金额: $ 29.1万
• 依托单位: PURDUE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-02-15 至 2010-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7216183
• 关键词: Abbreviations; Actins; Actomyosin; Address; Adhesions; Affect; Antibodies; Aplysia; Behavior; Biochemical; Biological Assay; Cell Adhesion Molecules; Cell Culture System; Cell Surface Receptors; Cell surface; Cells; Complementary DNA; Concanavalin A; Coupling; Cytoskeleton; DNA Sequence Rearrangement; Data; Enzymes; Event; F-Actin; Family; Fluorescence; Glycosylphosphatidylinositols; Goals; Growth; Growth Cones; Image; Imaging Techniques; Immunoglobulins; In Vitro; Injury; Life; Localized; Malignant Neoplasms; Mediating; Micromanipulation; Microscopy; Microtubules; Modeling; Molecular; Movement; Myosin ATPase; Neoplasm Metastasis; Nerve Regeneration; Neuraxis; Neurodegenerative Disorders; Neurons; Numbers; Peripheral; Phase; Phosphotransferases; Phosphotyrosine; Play; Principal Investigator; Process; Protein Dynamics; Protein Tyrosine Kinase; Race; Regulation; Research; Resolution; Role; Seawater; Sensory; Sequence Analysis; Signal Transduction; Site; Techniques; Testing; Time; Work; advanced system; attenuation; axon growth; axon guidance; cell motility; cellular imaging; enhanced green fluorescent protein; extracellular; improved; insight; mutant; neoplastic cell; neuronal growth; novel; programs; receptor; src-Family Kinases; tool

Understanding the mechanisms of neuronal growth cone guidance and motility is imperative, if we want to develop successful strategies for nerve regeneration after injury and neurodegenerative diseases. Although a large number of axon guidance molecules have been characterized in recent years, there are significant gaps in our understanding of the molecular and cellular mechanisms that the growth cone uses to integrate its sensory, signaling and motile functions. We have recently provided evidence that the immunoglobulin superfamily cell adhesion molecule apCAM mediates growth cone steering by substrate-cytoskeletal coupling. pCAM-actin coupling depends on Src kinase activity and results in actin flow attenuation ollowed by microtubule extension. Recent findings further suggest that microtubules influence Src kinase activity at adhesion sites. The goal of this project is to test the following hypothesis: Src kinase activity and microtubule dynamics regulate apCAM-cytoskeletal coupling in neuronal growth cone steering. Using the well-established high-resolution Aplysia growth cone system, advanced live cell imaging techniques, and a new set of molecular tools for Src tyrosine kinases, we will address three Specific Aims: (1) to determine if microtubules play a role early during apCAM-mediated growth cone steering. We will achieve this goal by combining a novel in vitro growth cone steering assay with microtubule fluorescent speckle microscopy to quantify the dynamic behavior of microtubules early before the major microtubule rearrangement occurs. The second Aim of this study is: (2) to identify Aplysia Src family kinases, an important group of tyrosine kinases implicated in the regulation of axonal growth, and to determine their subcellular localization, activation state and dynamics in growth cones. To achieve this goal, we will prepare antibodies and EGFP-fusion constructs of newly identified Src kinases in Aplysia. The third Aim is: (3) to determine the role of these Src family kinases in apCAM-mediated growth cone steering. Therefore, we will image Src-EGFP protein dynamics during growth cone steering events and test the effect of active and inactive Src mutants on apCAM-actin coupling and growth cone guidance. These studies will not only provide new insights into the role of microtubules and Src kinases in growth cone steering, but also unprecedented information on the dynamic behavior of this key signaling enzyme within a living neuron. Thus, they will have an impact on our understanding of axon guidance and nerve regeneration, as well as of tumor cell metastasis, another motile process, in which Src has been implicated.

如果我们想要为损伤和神经退行性疾病后的神经再生制定成功的策略，了解神经元生长锥引导和运动的机制是必不可少的。尽管近年来已经发现了大量轴突导向分子，但对于生长锥用于整合其感觉、信号和运动功能的分子和细胞机制，我们的理解还存在很大差距。我们最近提供的证据表明，免疫球蛋白超家族细胞黏附分子apCAM通过底物-细胞骨架偶联介导生长锥的导向。PCAM-肌动蛋白偶联依赖于Src激酶的活性，并导致微管伸展导致肌动蛋白流动减弱。最近的研究结果进一步表明，微管影响黏附部位的Src激酶活性。本项目的目的是验证以下假设：SRC激酶活性和微管动力学调节神经元生长锥体导向中的apCAM-细胞骨架偶联。 利用成熟的高分辨率海兔生长锥系统、先进的活细胞成像技术和一套新的Src酪氨酸激酶分子工具，我们将解决三个具体目标： (1)确定微管在apCAM介导的生长锥引导过程中是否起早期作用。我们将通过将一种新的体外生长锥形转向试验与微管荧光散斑显微镜相结合来实现这一目标，以在主要微管重排发生之前早期量化微管的动态行为。本研究的第二个目的是：(2)鉴定与轴突生长调控有关的一组重要的酪氨酸激酶，并确定它们在生长锥体中的亚细胞定位、激活状态和动态。为了实现这一目标，我们将制备抗体和新近在海兔中发现的Src激酶的EGFP融合构建体。第三个目的是：(3)确定这些Src家族蛋白在apCAM介导的生长锥体调控中的作用。因此，我们将 成像生长锥导向事件期间的Src-EGFP蛋白动态，并测试活跃和非活跃的Src突变体对apCAM-肌动蛋白偶联和生长锥导向的影响。这些研究不仅将为微管和Src激酶在生长锥调控中的作用提供新的见解，而且还将提供关于这一关键信号酶在活神经元中的动态行为的前所未有的信息。 因此，它们将影响我们对轴突引导和神经再生的理解，以及对肿瘤细胞转移的理解，这是另一个与Src有关的运动过程。