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.

如果我们想在损伤和神经退行性疾病后制定成功的神经再生策略，那么了解神经元生长锥引导和运动的机制至关重要。尽管近年来已经表征了大量的轴突引导分子，但我们对生长锥体用来整合其感觉，信号传导和运动功能的分子和细胞机制的理解存在很大的差距。我们最近提供了证据表明，免疫球蛋白超家族细胞粘附分子apcam介导了底物 - 骨骼骨骼偶联的生长锥转向。 PCAM-肌动蛋白偶联取决于SRC激酶活性，并导致微管延伸稳定的肌动蛋白流量衰减。最近的发现进一步表明，微管影响SRC激酶在粘附部位的活性。该项目的目的是检验以下假设：SRC激酶活性和微管动力学调节神经元生长锥转向中的apcam-骨骨骼偶联。 使用良好的高分辨率Aplysia生长锥系统，先进的活细胞成像技术以及一套新的SRC酪氨酸激酶分子工具，我们将解决三个特定的目标： （1）确定微管在APCAM介导的生长锥转向过程中是否起作用。我们将通过将新型的体外生长锥转向测定法与微管荧光斑点显微镜相结合，以量化主要微管重排之前的微管的动态行为。这项研究的第二个目的是：（2）鉴定Aplysia src家族激酶，这是一组重要的酪氨酸激酶，涉及轴突生长的调节，并确定其亚细胞定位，激活状态和生长锥中的动态。为了实现这一目标，我们将准备新鉴定的Aplysia中新鉴定的SRC激酶的抗体和EGFP融合构建体。第三个目的是：（3）确定这些SRC家族激酶在APCAM介导的生长锥转向中的作用。因此，我们会的 图像SRC-EGFP蛋白动力学在生长锥转向事件过程中，并测试活性和非活性SRC突变体对APCAM-ACTIN耦合和生长锥引导的影响。这些研究不仅将提供有关微管和SRC激酶在生长锥转向中的作用的新见解，而且还将提供有关这种关键信号酶在活神经元内的动态行为的前所未有的信息。 因此，它们将对我们对轴突引导和神经再生以及肿瘤细胞转移的理解产生影响，这是另一种涉及SRC的运动过程。