Mechanistic analysis of microtubule dynamics and stability in neurons

项目总结/摘要 神经元和它们的目标之间的通信依赖于适当的突触生长和活动。的 微管细胞骨架在突触终末发育中起核心作用，微管功能障碍是 与许多神经系统疾病有关。神经元包含稳定和动态的微管，这两个 突触的数量必须适当平衡，才能生长并形成稳定的连接。在本提案中，我们 使用体内遗传分析和无细胞体外生物物理方法的协同组合， 阐明微管动力学和稳定性平衡的机制。我们利用一种新的α- 微管蛋白突变体，改变正常微管平衡并扰乱突触生长。这种微管蛋白突变 破坏高度保守的、必需的乙酰化α-微管蛋白位点。翻译后修饰（PTM）， 例如乙酰化，具有直接和特异性调节微管稳定性和动力学的潜力， 形状突触形态发生，但相对较少的微管PTM已被研究。我们的初步数据 暗示这一先前未被表征的α-微管蛋白位点在调节微管蛋白二聚体的添加以促进细胞生长中起作用。 微管末端，这表明一种新的乙酰化为基础的机制来控制微管动力学。 基于我们的初步发现，我们将测试微管动力学和稳定性是 通过α-微管蛋白乙酰化和其他已知的调节剂来平衡突触终末形态发生（Aim 1）。我们将以果蝇的神经肌肉接头为模型，研究操纵 微管动力学和稳定性对两种不同的运动神经元类型，称为Ib型和Is型，其 突触末梢具有不同的形态和传输特性。我们的初步数据显示， 改变微管细胞骨架对Ib型和Is型突触的生长具有显著不同的影响。 terminals.我们将测试这一假设，即稳定和动态微管是唯一的平衡，在不同的 神经元类型，以建立不同的神经元特异性突触结构和活动（目的2）。结合起来，我们的 研究将揭示调节突触微管网络的新机制，并提供基本的 对微管在创造不同突触形态和功能中所起的核心作用的新见解。

PROJECT SUMMARY/ABSTRACT Communication between neurons and their targets depends on proper synaptic growth and activity. The microtubule cytoskeleton plays a central role in synaptic terminal development, and microtubule dysfunction is associated with many neurological disorders. Neurons contain stable and dynamic microtubules, and these two populations must be properly balanced for synapses to grow and form stable connections. In this proposal, we use a synergistic combination of in vivo genetic analyses and cell-free in vitro biophysical approaches to elucidate the mechanisms by which microtubule dynamics and stability are balanced. We leverage a novel α- tubulin mutant that alters the normal microtubule balance and perturbs synaptic growth. This tubulin mutation disrupts a highly conserved, essential α-tubulin site that is acetylated. Post-translational modifications (PTMs), such as acetylation, have the potential to directly and specifically regulate microtubule stability and dynamics to shape synaptic morphogenesis, yet relatively few microtubule PTMs have been studied. Our preliminary data implicate this previously uncharacterized α-tubulin site in regulating the addition of tubulin dimers to growing microtubule ends, which suggests a novel acetylation-based mechanism to control microtubule dynamics. Based on our preliminary findings, we will test the hypothesis that microtubule dynamics and stability are balanced by α-tubulin acetylation and other known regulators to shape synaptic terminal morphogenesis (Aim 1). We will use the Drosophila neuromuscular junction as a model and investigate the effects of manipulating microtubule dynamics and stability on two different motor neuron types, called type Ib and type Is, whose synaptic terminals have distinct morphologies and transmission properties. Our preliminary data indicate that altering the microtubule cytoskeleton has strikingly different effects on the growth of type Ib and Is synaptic terminals. We will test the hypothesis that stable and dynamic microtubules are uniquely balanced in different neuron types to establish distinct neuron-specific synaptic structures and activities (Aim 2). Combined, our studies will reveal novel mechanisms that regulate synaptic microtubule networks and provide fundamental new insight into the central role that microtubules play in creating diverse synaptic morphologies and functions.