Spatiotemporal Control of Cofilin Activity During Growth Cone Migration

生长锥迁移过程中丝切蛋白活性的时空控制

• 批准号: 8255064
• 负责人: Eric A Vitriol
• 金额: $ 5.13万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-12-01 至 2013-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8255064
• 关键词: Actins; Address; Axon; Behavior; Biological Assay; Biological Process; Cell physiology; Cells; Complex; Conflict (Psychology); Cytoskeleton; Defect; Disease; Ensure; Event; Failure; Filament; Financial compensation; Genetic; Growth Cones; Hydrolysis; Label; Lasers; Lead; Life; Light; Mediating; Methodology; Microfilaments; Microscopy; Molecular; Monitor; Morphology; Natural regeneration; Nerve; Nerve Regeneration; Neurons; Outcome; Output; Photosensitizing Agents; Process; Proteins; Quantitative Microscopy; Recruitment Activity; Regulation; Reporting; Research; Resolution; Role; Signal Pathway; Spinal; Structure; Techniques; Testing; Time; Up-Regulation; Xenopus; Xenopus laevis; axon growth; axon guidance; cell motility; cell type; chromophore; cofilin; design; developmental disease; genetic regulatory protein; injured; inorganic phosphate; insight; irradiation; loss of function; migration; monomer; polymerization; prevent; response; spatiotemporal; tool

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.

描述（由申请人提供）：数十年的研究提供了许多关于Cofilin如何与肌动蛋白相互作用并改变肌动蛋白的见解。然而，这些动作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，同样的活动可能导致相反的结果。例如，已显示Cofilin的局部上调与吸引性和排斥性生长锥引导相关。这些看似矛盾的结果很可能是由Cofilin在何处、何时以及多少被激活所介导的。几项研究表明，Cofilin激活的定位和时间对于确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有限。我们假设，一种方法，使我们能够立即Cofilin与亚细胞精度将导致发现新的机制信息，如何Cofilin的功能，以调节肌动蛋白网络和控制生长锥运动。为了确定生长锥迁移过程中Cofilin活性的时空作用，我们提出了以下具体目标：（1）开发Cofilin局部和瞬时失活的方法;（2）确定Cofilin瞬时失活对生长锥中肌动蛋白分布和动力学的影响;（3）确定Cofilin局部失活对生长锥运动性的影响。使用一种名为生色团辅助激光灭活（卡利）的技术，我们将证明我们能够以亚细胞精度立即抑制Cofilin。我们将开发这种方法，使其成为其他实验室想要确定局部Cofilin失活的功能后果的普遍适用和有用的工具。通过将卡利与高分辨率活细胞显微镜相结合，我们将在瞬时Cofilin耗尽后真实的时间内监测肌动蛋白网络的变化。最后，我们将使用卡利来确定Cofilin的局部失活如何影响生长锥迁移和引导。轴突引导的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，但其复杂的功能作用尚未完全阐明。在这项提案中，我们将揭示有关Cofilin如何调节轴突生长的新机制信息，以便更好地理解其在指导相关疾病中的作用。