Molecular determinants and function of axonal actin assemblies

本申请的总体目标是阐明神秘的轴突肌动蛋白的分子组织和功能。 我的实验室最近发现的组件。肌动蛋白沿轴突轴突的组织相对被忽视了 几十年，可能是因为常规染色-使用经典的肌动蛋白细丝标记-只显示了 零星的、无趣的图案。然而，我们和其他人最近使用超分辨率显微镜进行的研究 微光成像揭示了轴突中肌动蛋白的隐藏世界--充满了精致的周向 质膜下有环状突起，沿轴突轴线快速伸长的线状细丝。 我们很幸运地站在了这些发现的前沿，我们的研究揭示了令人着迷的 轴突中的肌动蛋白动力学。具体地说，我们发现轴突的焦点“热点”是肌动蛋白，间隔约3-4微米 -肌动蛋白不断聚合和解聚。这些热点产生了长长的肌动蛋白细丝 沿着轴突快速(双向)伸长(我们称之为“肌动蛋白痕迹”)。肌动蛋白环、踪迹和 在活体轴突中可以看到热点，我们在这个方案中也将使用体内模型系统。尽管 已经看到了整体组装，分子事件是这些分子产生和维持的基础 结构尚不清楚。重要的是，它们在轴突中的功能作用尚不清楚。在这里，我们提出三个具体的建议 旨在阐明这些新发现的肌动蛋白组件的组织和功能：目标1：确定 在轴突中启动肌动蛋白拖尾的机制。目标2：确定延长轴突中肌动蛋白踪迹的机制。目标 #3：确定轴突肌动蛋白组件的功能。

The overall goal of this application is to clarify the molecular organization and function of enigmatic axonal actin assemblies that my lab recently discovered. Actin organization along axon shafts has been relatively ignored for decades, probably because routine staining – using the classic actin-filament marker phalloidin – only shows a patchy, uninteresting pattern. However, recent studies from us and others' using super-resolution microscopy and low-light imaging have revealed a hidden world of actin in axon shafts – replete with elaborate circumferential rings underneath the plasma membrane, and rapidly elongating linear filaments along the axis of the axon shaft. We have been fortunate to be at the forefront of these discoveries, and our studies have revealed fascinating actin dynamics in axons. Specifically, we found that axons have focal “hotspots” of actin – spaced ~ 3-4 µm apart – where actin continuously polymerizes and depolymerizes. These hotspots give rise to long actin filaments that rapidly (and bidirectionally) elongate along the axon shaft (we named these “actin trails”). Actin rings, trails and hotspots are seen in axons in vivo, and we will use in vivo model systems in this proposal as well. Although the overall assemblies have been seen, molecular events underlying the generation and maintenance of these structures is unclear. Importantly, their functional roles in axons are unknown. Here we propose three specific aims to clarify the organization and function of these newly-discovered actin assemblies: Aim #1: Identify mechanisms initiating actin trails in axons. Aim #2: Identify mechanisms elongating actin trails in axons. Aim #3: Determine functions of axonal actin assemblies.