Spatiotemporal Dynamics of RhoA Activation in Growth Cone Motility

描述(申请人提供)：小的GTP酶RhoA导致生长锥体塌陷和停止轴突生长，从而抑制受损神经的再生。相反，RhoA也被证明在特定情况下促进生长锥前进，并对指导生长锥运动很重要。这项建议的广泛目标是推断，通过精确控制RhoA激活动力学和定位，该蛋白如何刺激相反的细胞行为。为了解决这一矛盾，我们将利用生物传感器直接可视化RhoA在生长和收缩神经元中的激活，并操纵微管来探索RhoA信号的潜在机制。具体目标1将使用新的图像处理工具来生成亚秒和亚微米分辨率的RhoA激活的动态地图，以确定生长锥体在伸展和坍塌过程中RhoA激活的时空动力学。微管细胞骨架是生长锥运动和导向的主要调节因子，也是RhoA的主要下游靶点和上游激活剂。具体目标2将确定RhoA激活与生长锥体中微管分布和动力学的关系。 与公共卫生相关：除了神经系统再生衰竭外，还有许多严重的、使人衰弱的发育疾病，轴突生长不能正常进行。了解生长锥体运动和轴突伸展的基本机制有助于开发治疗神经系统损伤和疾病的新方法。

DESCRIPTION (provided by applicant): The small GTPase RhoA causes growth cone collapse and halts axon growth, thereby inhibiting regeneration of injured nerves. In contrast, RhoA has also been shown to promote growth cone advance under specific circumstances, and is important for guidance in growth cone motility. The broad objective of this proposal is to deduce how, through precise control of RhoA activation kinetics and localization, this protein can stimulate opposite cell behaviors. To resolve this conflict, we will utilize a biosensor to directly visualize the activation of RhoA in growing and retracting neurons, and manipulate microtubules to probe underlying mechanisms in RhoA signaling. Specific Aim 1 will determine the spatiotemporal dynamics of RhoA activation in growth cones during both extension and collapse, using novel image processing tools to generate a dynamic map of RhoA activation with subsecond and submicron resolution. The microtubule cytoskeleton is a master regulator of growth cone motility and guidance; it is both a major downstream target and upstream activator of RhoA. Specific Aim 2 will determine the relationship between RhoA activation and microtubule distribution and dynamics in growth cones. PUBLIC HEALTH RELEVANCE: In addition to nervous system regeneration failure, there are a host of severe and debilitating developmental diseases in which axon growth fails. Understanding the basic mechanism behind growth cone motility and axon extension can lead to the development of new treatments for nervous system injury and disease.