Mechanistic analysis of microtubule dynamics and stability in neurons

项目摘要/摘要 神经元和它们的目标之间的交流依赖于适当的突触生长和活动。这个 微管细胞骨架在突触终末发育中起核心作用，而微管功能障碍 与许多神经疾病有关。神经元含有稳定和动态的微管，这两个 种群必须适当地平衡，才能使突触生长并形成稳定的连接。在这项提案中，我们 使用体内遗传分析和体外无细胞生物物理方法的协同组合来 阐明微管动力学和稳定性平衡的机制。我们利用一种新颖的α- 微管蛋白突变体，改变正常的微管平衡，扰乱突触生长。这种微管蛋白突变 破坏高度保守的、基本的α-微管蛋白乙酰化位点。翻译后修改(PTM)， 例如乙酰化，有可能直接和特异性地调节微管稳定性和动力学，从而 形状突触的形态发生，但微管PTM的研究相对较少。我们的初步数据 该未知的α-微管蛋白位点参与调节微管蛋白二聚体在生长过程中的添加 微管结束，这表明了一种新的基于乙酰化的机制来控制微管的动力学。 基于我们的初步发现，我们将检验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因子来平衡突触终末形态发生(AIM 1)。我们将以果蝇神经肌肉接头为模型，研究手法的效果。 微管在两种不同类型运动神经元上的动力学和稳定性，称为Ib型和I型，其 突触终末具有不同的形态和传递特性。我们的初步数据显示 改变微管细胞骨架对Ib型突触的生长有明显不同的影响 终点站。我们将检验这样的假设，即稳定的和动态的微管在不同的 建立独特的神经元特有的突触结构和活动的神经元类型(目标2)。加在一起，我们的 研究将揭示调节突触微管网络的新机制，并为 对微管在创造不同的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.