Using cryo-electron tomography and live-cell fluorescent imaging to study the role of cofilin in regulating neuronal filopodial structure and dynamics

项目摘要/摘要 神经元如何将它们的过程引导到正确的结合伙伴是一项复杂的任务，但在 发展和受伤后的恢复。它涉及许多细胞骨架蛋白的高度协调作用。 和他们在生长锥体内的结合伙伴，在延伸的轴突的尖端，当他们摸索着穿过 神经针。关于调节轴突生长和转向的信号通路，我们知道得很多，但是 关于分子结构如何结合在一起实现生长锥体行为的细节仍不清楚。这 该项目最初将重点放在原位捆绑的结丝状肌动蛋白细丝(Cofilin修饰的F-肌动蛋白)的结构上，以及 这种新的细丝结构和束蛋白交联物是如何决定细丝的动力学的。在这里，我们建议 丝状轴的行为部分地受从束蛋白交联物到交联物交联物的转变所支配 花丝，这使得丝轴维管束更具柔韧性。实验围绕三个目标展开：1)研究 筋膜蛋白和粘连蛋白连接的肌动蛋白束的高分辨结构以确定它们对肌动蛋白的影响 结构，2)确定筋膜蛋白和粘附素浓度的变化如何调节丝状体动力学和 结构，以及3)确定LIMKI和SSH1如何形成双向监管机制的核心 通过调节粘附素上Ser3的磷酸化状态来调节肌动蛋白的结构。

Project Summary/Abstract How neurons guide their processes to the correct binding partner is a complicated task, but is critical during development and recovery from injury. It involves the highly coordinated action of many cytoskeletal proteins and their binding partners within the growth cone at the tips of extending neurites, as they feel their way through the neuropil. There is a lot known about the signaling pathways that regulate neurite outgrowth and turning, but the details of how molecular structures come together to achieve growth cone behavior are still unclear. This project will initially focus on the structure of bundled cofilactin filaments (cofilin-decorated F-actin) in situ, and how this novel filament structure and fascin cross-linking determine filopodial dynamics. Here we propose that filopodial behavior is governed partially by the transition from the fascin cross-linked to a cofilin cross-linked filaments, that makes filopodial bundles more pliable. Experiments are focused around three aims: 1) to study the high-resolution structure of fascin- and cofilin-linked actin bundles to determine their impact on actin structure, 2) to determine how changes in fascin and cofilin concentration regulate filopodial dynamics and structure, and 3) determine how LIMKI and SSH1 form the core of a bidirectional regulatory mechanism for regulating actin architecture via tuning the phosphorylation state of Ser3 on cofilin.