ROLE OF MYOSIN ISOFORMS IN NERVE GROWTH CONE MOTILITY

描述(调查人员摘要)：这项工作的长期目标 建议理解生长锥的分子机制 发育中的神经元的运动性。轴突由细胞外线索引导 通过局部影响细胞骨架来指导细丝运动 动力学。了解这一现象的分子基础需要 了解神经回路是如何在胚胎发育和 以确定哪些因素会导致神经性出生缺陷。 此外，这些分子机制很可能用于神经 再生和对它们的理解将有助于设计 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 已经确定了可能参与转导的候选蛋白质 丝状运动和引导，但在体内建立它们的功能 在神经元生长锥体中一直是困难的。长期目标 将通过应用微尺度发色团辅助激光来解决 灭活(微卡)，本实验室开发的一种方法，以 以前所未有的水平灭活特定的细胞内蛋白质 空间和时间分辨率。卡利经过了严格的测试， 用于确定膜蛋白在神经细胞中的活体作用 发展。使用这项技术来解决 生长锥运动的分子机制。 具体地说，talin、vinculin、pp60c-src和pp60c-src的体内作用 肌球蛋白在细丝运动中的作用将通过局部灭活来确定 它们在生长锥体中，并通过视频观察结果- 增强显微镜和定量形态计量学。实验也是 建议询问这些蛋白质是否在底物介导的过程中发挥作用 当生长锥体到达图案化的边界时使用微卡利进行引导 底物。这些研究将使用鸡背根神经节进行 培养中的神经元，一个具有良好特性和可操控的系统 存在针对许多蛋白质的抗体，这些蛋白质可能是 参与生长锥运动。建议的实验重点是 因为我们的初步实验结合了 体外生化数据表明了它们如何发挥作用和 在丝状伪足的延伸和调节中相互作用。微卡利将 通过使这些蛋白质失活来测试这一模型 组合给出特定的表型，支持或驳斥 建议的互动。

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.