Regulation of Neuronal Motility: the role of actin filament turnover

描述（由申请人提供）：神经元是已知极化程度最高的细胞。肌动蛋白丝和微管细胞骨架蛋白的组装和拆卸是神经元极性产生和神经元生长调节的基础。包括荧光散斑显微镜（FSM）在内的成像技术的最新发展使细胞骨架聚合物在活细胞中的组装、拆卸和易位能够直接可视化。自动荧光特征跟踪的新算法允许在图像范围内定量分析蛋白质运动，从而表征细胞骨架系统的反应。这些计算上的进步也为神经元运动事件的数学建模铺平了道路。我们一直在使用这些技术来表征参与神经元生长的肌动蛋白丝和微管蛋白动力学。我们的重点是位于发展中的轴突和树突过程远端的高度运动结构，称为生长锥。生长锥通过对环境中扩散和/或结合的分子信号作出反应来调节神经突发育的速度和方向。线索识别进入信号转导途径，最终影响最终的细胞骨架蛋白效应器。逆行肌动蛋白流动是生长锥运动的一个普遍方面。这是由于前缘不断的肌动蛋白网络组装加上生长锥更中心的T区肌凝蛋白II的收缩而导致的后向网络“拉动”。最近的许多工作集中在协调维持逆行血流所需的前沿肌动蛋白丝组装的蛋白质上。然而，肌动蛋白丝的周转也是维持逆行流动的稳态聚合物通量特性所必需的。该项目旨在解决肌动蛋白周转和神经生长锥网络循环的问题，因为这一重要功能尚不清楚。我们的动机是我们最近的发现，局部肌凝蛋白II收缩在包括丝状足的极化肌动蛋白束的稳定周转中起着意想不到的作用。这些肌动蛋白束在前缘组装，然后通过逆行流运输到T区，在那里它们经历负端切断和翻转。我们的研究表明，局部肌凝蛋白II的收缩力增强了一个单独的肌动蛋白丝切断因子的活性。我们建议研究cofilin作为肌动蛋白束回收的候选物，并在其他地方表征cofilin的功能。虽然cofilin与生长锥功能有关，但其在细胞中的作用机制尚不清楚；事实上，最近的生物化学证据表明，cofilin的作用是复杂的，并且依赖于浓度。为了解决这个突出的问题，新的分子成像分析将用于将肌动蛋白丝周转率与活生长锥中肌动蛋白丝-cofilin相互作用直接联系起来。我们还将研究cofilin在允许细胞粘附分子底物刺激的急性生长锥推进的情况下的功能。公共卫生相关性：神经生长锥体运动对于轴突生长和发育过程中的引导以及神经损伤后的再生是必要的。生长锥的运动取决于肌动蛋白丝网络的稳定组装和拆卸。相关的肌动蛋白网络是如何以及在哪里组装的相对来说已经很好理解了；然而，网络的拆解过程是一个谜。这个项目解决了这个突出的问题。

DESCRIPTION (provided by applicant): Neurons are the most polarized cells known. Regulated assembly and disassembly of actin filament and microtubule cytoskeletal proteins is fundamental to generation of neuronal polarity and regulation of neuronal growth. Recent developments in imaging technology including fluorescent speckle microscopy (FSM) enable direct visualization of cytoskeletal polymer assembly, disassembly and translocation of in living cells. New algorithms for automated fluorescent feature tracking allow quantitative analysis of protein movements on an image-wide basis, allowing characterization of cytoskeletal system responses. These computational advances also pave the way for mathematical modeling of neuronal motility events. We have been using these techniques to characterize the actin filament and microtubule protein dynamics involved in neuronal growth. Our focus has been on the highly motile structure located at the distal end of developing axonal and dendritic processes called the growth cone. Growth cones regulate the rate and direction of neurite advance by responding to diffusible and/or bound molecular cues in their environment. Cue recognition feeds into signal transduction pathways that ultimately impinge on the final cytoskeletal protein effectors. Retrograde actin flow is a ubiquitous aspect of growth cone motility. It results from constant actin network assembly at the leading edge plus rearward network "pulling" due to myosin II contractility in a more central region of the growth cone called the T zone. Much recent work has focused on proteins that coordinate the actin filament assembly at the leading edge necessary to sustain retrograde flow. However, actin filament turnover is also necessary to maintain the steady state polymer flux characteristic of retrograde flow. This project is aimed at addressing the problem of actin turnover and network recycling in neuronal growth cones since this important function is not understood. We are motivated by our recent discovery that localized myosin II contractility plays an unexpected role in the steady turnover of polarized actin bundles that comprise filopodia. These actin bundles are assembled at the leading edge, and then transported by retrograde flow into the T zone where they undergo minus end severing and turnover. Our studies suggest that localized myosin II contractility potentiates the activity of a separate actin filament severing factor. We propose to investigate cofilin as an actin bundle recycling candidate and characterize cofilin function elsewhere as well. Although cofilin has been implicated in growth cone function, its mechanism of action in cells is not well understood; indeed, recent biochemical evidence suggests cofilin actions are complex and concentration dependent. To address this outstanding problem, novel molecular imaging assays will be used to correlate actin filament turnover rates directly with actin filament-cofilin interactions in living growth cones. We will also look at cofilin function in the context of acute growth cone advance stimulated by application of permissive cell adhesion molecule substrates. PUBLIC HEALTH RELEVANCE: Neuronal growth cone motility is necessary for axon growth and guidance during development and for regeneration after nerve injury. Growth cone motility depends on the steady assembly and disassembly of actin filament networks. How and where the relevant actin networks assemble is relatively well understood; however, the network disassembly process is a mystery. This project addresses this outstanding problem.