ROLE OF MYOSIN ISOFORMS IN NERVE GROWTH CONE MOTILITY

描述（研究者摘要）：长期目标 建议是了解生长锥的分子机制 发育中的神经元的运动性。轴突由细胞外信号引导 通过局部影响细胞骨架来指导丝状伪足运动 动力学了解其分子基础是必要的， 了解胚胎发育过程中神经回路是如何形成的， 以确定哪些因素会导致神经性出生缺陷。 此外，这些分子机制可能用于神经 再生和理解他们将有助于设计 神经损伤后的治疗 在神经发育、细胞生物学和信号方面的大量工作 转导已经确定了可能参与 丝状伪足运动和引导，但建立其体内功能 在神经生长锥中的作用是很困难的。长期目标 将通过应用微尺度发色团辅助激光来解决 灭活（micro-CALI）是本实验室开发的一种方法， 具有前所未有水平的细胞内特异性蛋白 空间和时间分辨率。卡利经过严格测试， 用于确定膜蛋白在神经细胞中的体内作用， 发展现在正是利用这种技术来解决 生长锥运动的分子机制。 具体地，研究了talin、黏着斑蛋白、pp 60 c-src和p53的体内作用。 丝状伪足运动中的肌球蛋白将通过局灶性灭活 将它们放入生长锥中，通过视频观察它们的行为， 增强显微镜和定量形态测定法。实验也 提出要问这些蛋白质是否在底物介导的 当生长锥到达图案化的边界时， 印刷受体.这些研究将使用鸡背根神经节进行 神经元的培养，一个良好的特点和操纵系统， 存在针对许多蛋白质的抗体， 与生长锥运动有关建议的实验重点是 因为我们的初步实验加上 体外生物化学数据表明了它们如何发挥作用的模型， 在丝状伪足的延伸和调节中相互作用。微CALI将 应用于测试这个模型，通过灭活这些蛋白质， 组合，以给出将支持或反驳的特定表型， 建议互动。

DESCRIPTION (Investigator's Abstract): The long term objective of this proposal is to understand the molecular mechanisms of growth cone motility in developing neurons. Axons are guided by extracellular cues that direct filopodial motility by locally affecting cytoskeletal dynamics. Understanding the molecular basis of this is required to understand how neurocircuitry is formed during embryonic development and to establish what factors can cause neurological birth defects. Moreover, these molecular mechanisms are likely to be used in nerve regeneration and an understanding of them will aid in designing treatment after nerve injury. Considerable work in neural development, cell biology, and signal transduction have identified candidate proteins that may be involved in filopodial motility and guidance but establishing their in vivo function in neuronal growth cones has been difficult. The long term objective will be addressed by applying microscale chromophore assisted laser inactivation (micro-CALI), a method developed in this laboratory, to inactivate specific intracellular proteins with an unprecedented level of spatial and temporal resolution. CALI has been rigorously tested and used to determine the in vivo roles of membrane proteins in neural development. It is timely to use this technique to address the molecular mechanisms of growth cone motility. Specificically, the in vivo roles of talin, vinculin, pp60c-src, and the myosins in filopodial motility will be determined by focally inactivating them in growth cones and observing the resulting behavior by video- enhanced microscopy and quantitative morphometry. Experiments are also proposed to ask if these proteins play a role in substrate-mediated guidance by using micro- CALI as growth cones reach borders on patterned substrates. These studies will be done using chick dorsal root ganglion neurons in culture, a well characterized and manipulatable system for which there exist antibodies against many proteins that are potentially involved in growth cone motility. The proposed experiments are focused on these proteins because our preliminary experiments coupled with in vitro biochemical data suggest a model for how they function and interact in the extension and regulation of filopodia. Micro-CALI will be applied to test this model by inactivating these proteins in combination to give specific phenotypes that will support or refute the proposed interactions.