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。肌动蛋白组织沿着轴突轴已被相对忽视， 几十年来，可能是因为常规染色-使用经典的肌动蛋白丝标记鬼笔环肽-只显示了一个 不完整的无趣的图案然而，最近我们和其他人使用超分辨率显微镜的研究表明， 低光成像揭示了轴突中隐藏的肌动蛋白世界--充满了精心设计的周向 质膜下的环，以及沿着轴突轴的轴沿着迅速伸长的线状细丝。 我们很幸运能站在这些发现的最前沿，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，我们发现轴突有肌动蛋白的焦点-间隔约3-4 μm - 其中肌动蛋白连续地聚合和解聚。这些热点产生长的肌动蛋白丝， 沿着沿着轴突轴迅速（双向）伸长（我们将这些称为“肌动蛋白轨迹”）。肌动蛋白环，轨迹和 在体内轴突中观察到热点，并且我们也将在该提议中使用体内模型系统。虽然 已经看到了整体组装，这些组装的产生和维持的分子事件 结构不清楚。重要的是，它们在轴突中的功能作用是未知的。在这里，我们提出三个具体的 目的是阐明这些新发现的肌动蛋白组装体的组织和功能：目的#1：识别 在轴突中启动肌动蛋白踪迹的机制。目的#2：确定轴突中延长肌动蛋白踪迹的机制。目的 #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.