Spatiotemporal Control of Cofilin Activity During Growth Cone Migration

描述（由申请人提供）：数十年的研究提供了关于 Cofilin 如何与肌动蛋白相互作用并改变肌动蛋白的大量见解。然而，这些作用在细胞中具有复杂的输出，包括促进肌动蛋白网络的组装和分解。因此，同样的活动可能会导致相反的结果。例如，Cofilin 的局部上调已被证明与有吸引力和排斥性生长锥引导有关。这些看似相互矛盾的结果很可能是由 Cofilin 的激活地点、时间和程度所调节的。多项研究表明，丝切蛋白激活的定位和时间对于确定其调用哪些下游行为至关重要。然而，直接测试这些想法的工具是有限的。我们假设，一种能够以亚细胞精度立即灭活 Cofilin 的方法将导致发现新的机制信息，即 Cofilin 如何发挥作用来调节肌动蛋白网络和控制生长锥运动。为了确定 Cofilin 活性在生长锥迁移过程中的时空作用，我们提出以下具体目标：（1）开发一种 Cofilin 局部瞬时失活的方法； (2)确定Cofilin瞬时失活对生长锥中肌动蛋白分布和动态的影响； (3)确定Cofilin局部失活对生长锥运动的影响。使用一种称为发色团辅助激光灭活 (CALI) 的技术，我们将证明我们能够以亚细胞精度立即灭活 Cofilin。我们将开发这种方法，使其成为其他想要确定局部 Cofilin 失活的功能后果的实验室的普遍适用且有用的工具。通过将 CALI 与高分辨率活细胞显微镜相结合，我们将在瞬时 Cofilin 耗尽后实时监测肌动蛋白网络的变化。最后，我们将使用 CALI 来确定 Cofilin 的局部失活如何影响生长锥迁移和引导。轴突引导缺陷与发育障碍和神经再生失败有关。了解轴突生长的基本生物过程将有助于设计更好、更有效的疾病治疗方法。 Cofilin 与生长锥运动有关，但其复杂的功能作用尚未完全阐明。在本提案中，我们将揭示有关 Cofilin 如何发挥调节轴突生长作用的新机制信息，以便更好地理解其在指导相关疾病中的作用。

DESCRIPTION (provided by applicant): Decades of research have provided numerous insights as to how Cofilin interacts with and alters actin. However, these actions have complex outputs in the cell, including promoting both assembly and breakdown of actin networks. Thus the same activity can lead to opposite outcomes. For example, the local upregulation of Cofilin has been shown to be both associated with attractive and repulsive growth cone guidance. It is likely that that these seemingly conflicting results are mediated by where, when, and how much Cofilin is being activated. Several studies have shown that localization and timing of Cofilin activation is critical in determining which downstream behaviors it invokes. However, the tools to directly test these ideas have been limited. We hypothesize that a methodology which allows us to instantly inactivate Cofilin with subcellular precision will lead to the discovery of new mechanistic information of how Cofilin functions to regulate actin networks and to control growth cone motility. To determine the spatiotemporal role of Cofilin activity during growth cone migration, we propose the following Specific Aims: (1) To develop a methodology for local and instantaneous inactivation of Cofilin; (2) to determine the effects of instantaneous inactivation of Cofilin on actin distribution and dynamics in growth cones; and (3) to determine the effects of local inactivation of Cofilin on growth cone motility. Using a technique called Chromophore Assisted Laser Inactivation (CALI), we will show that we are able to instantly inactivate Cofilin with subcellular precision. We will develop this methodology so that it will be a generally applicable and useful tool for other labs who want to determine the functional consequences of local Cofilin inactivation. By combining CALI with high resolution live cell microscopy, we will monitor actin network changes in real time after instantaneous Cofilin depletion. Finally, we will use CALI to determine how local inactivation of Cofilin effects growth cone migration and guidance. Defects in axon guidance are associated with developmental disorders and nerve regeneration failure. Understanding the fundamental biological processes that underlie axon growth will allow for the design of better, more effective disease treatments. Cofilin has been implicated in growth cone motility, but its complex functional role has yet to be fully elucidated. In this proposal, we will uncover new mechanistic information about how Cofilin functions to regulate axon growth so that its role in guidance related disorders can be better understood.