Regulation of Neuronal Growth Cone Guidance

了解神经生长锥引导和运动的机制是必要的，如果我们想开发成功的策略，损伤和神经退行性疾病后的神经再生。虽然近年来已经表征了大量轴突引导分子，但是在我们对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的理解中存在显著的差距。我们最近提供的证据表明，免疫球蛋白超家族细胞粘附分子apCAM介导生长锥转向基板细胞骨架耦合。 pCAM-actin偶联依赖于Src激酶活性，并导致微管延伸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Src激酶活性的粘附网站。本项目的目标是测试以下假设：Src激酶活性和微管动力学调节apCAM细胞骨架耦合神经生长锥转向。 使用完善的高分辨率非洲龙属生长锥系统，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，我们将解决三个具体目标： (1)以确定微管是否在apCAM介导的生长锥转向的早期发挥作用。我们将通过结合一种新的体外生长锥转向试验与微管荧光斑点显微镜来实现这一目标，以量化微管的动态行为早期之前，主要的微管重排发生。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-Astrasia Src家族激酶，并确定它们在生长锥中的亚细胞定位、激活状态和动力学。为了实现这一目标，我们将制备抗体和EGFP融合构建体的新确定的Src激酶在Astrasia。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。所以我们会 在生长锥转向事件期间成像Src-EGFP蛋白动力学，并测试活性和非活性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激酶在生长锥转向中的作用提供新的见解，而且还将为活神经元内这种关键信号酶的动态行为提供前所未有的信息。 因此，它们将对我们理解轴突导向和神经再生以及肿瘤细胞转移产生影响，这是另一个涉及Src的能动过程。

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.