Regulation of Neuronal Growth Cone Guidance

如果我们想要在损伤和神经退行性疾病后制定成功的神经再生策略，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的轴突引导分子，但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仍存在重大差距。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通过底物-细胞骨架偶联介导生长锥转向。pcam -肌动蛋白偶联依赖于Src激酶活性，导致肌动蛋白流动衰减，随后微管延伸。最近的研究结果进一步表明，微管影响粘附位点的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apcam -细胞骨架耦合在神经元生长锥转向。

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.