Spatiotemporal Control of Cofilin Activity During Growth Cone Migration

描述(由申请人提供)：数十年的研究已经提供了大量关于cofilin如何与肌动蛋白相互作用和改变肌动蛋白的见解。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具有吸引力和排斥性的生长锥引导有关。这些看似相互矛盾的结果很可能是由何时、何地和多少粘连蛋白被激活所调节的。一些研究表明，cofilin激活的局部化和时机对于决定它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一直有限。我们假设，一种方法，使我们能够立即以亚细胞精度灭活cofilin，将导致新的机制信息的发现，cofilin如何作用于调节肌动蛋白网络和控制生长锥体运动。为了确定Cofilin活性在生长锥迁移过程中的时空作用，我们提出了以下具体目标：(1)建立Cofilin局部和瞬时失活的方法学；(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。使用一种名为发色团辅助激光灭活(CALI)的技术，我们将展示我们能够以亚细胞精度立即灭活粘连蛋白。我们将开发这一方法，以便它将成为其他希望确定局部cofilin失活的功能后果的实验室普遍适用和有用的工具。通过结合CALI和高分辨率活细胞显微镜，我们将实时监测瞬时cofilin耗尽后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学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，但其复杂的功能尚未完全阐明。在这项提案中，我们将揭示有关cofilin如何调节轴突生长的新的机械性信息，以便更好地了解它在指导相关障碍中的作用。

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.