Single molecule analysis of cytoskeletal cross-linking proteins

描述(申请人提供)：发展神经科学的关键问题之一是了解不同类型的神经元如何对外部信号做出反应，以建立与其适当目标的突触，并产生其特定的轴突和树突结构。由肌动蛋白细丝和微管组成的细胞骨架网络作为一个脚手架，允许导航的生长锥改变它们的轨迹，以响应吸引和排斥的引导提示，定义成熟神经元的形态，并对损伤做出反应。为了让所有这些过程正常进行，神经元必须以高度调控的方式协调肌动蛋白细丝和微管的动态。这项建议的目标是通过研究果蝇短停(Shot)来了解神经元细胞骨架动力学是如何调节的。缺乏射击的苍蝇表现出轴突生长缺陷和中枢神经系统畸形。斑点蛋白在动物谱系中是保守的，它们的突变会导致小鼠的感觉神经元退化和发育性脑畸形，以及人类的神经退行性疾病。在细胞水平上，镜面蛋白充当交联剂，在物理上连接肌动蛋白和微管。尽管神经系统具有这些基本功能，但我们对幽灵蛋白如何在分子水平上进行调控知之甚少。在这个提案中，我们将使用Shot作为一个范例，来研究如何使用尖端的单分子可视化技术来调节镜像板。1)我们将建立一种在单分子分辨率下可视化显示快照肌动蛋白-微管交联的方法。2)我们将测试 假设Shot是由分子内抑制机制调节的。鉴于这一蛋白家族成员之间的高度保守性，我们预计这些调节机制也将在脊椎动物镜板蛋白中保守。

DESCRIPTION (provided by applicant): One of the key problems in developmental neuroscience is understanding how different types of neurons respond to external signals to establish synapses with their appropriate targets and to generate their specific axonal and dendritic architecture. The cytoskeletal network of actin filaments and microtubules acts as a scaffolding to allow navigating growth cones to alter their trajectories in response to attractive and repulsive guidance cues, to define the morphology of mature neurons, and to remodel in response to injury. For all of these processes to proceed normally, neurons must coordinate the dynamics of actin filaments and microtubules in a highly regulated manner. The goal of this proposal is to understand how neuronal cytoskeletal dynamics are regulated by studying the Drosophila spectraplakin Short stop (Shot). Flies lacking Shot exhibit axon outgrowth defects and CNS malformation. Spectraplakins are conserved among animal lineages and their mutation causes sensory neuron degeneration and developmental brain malformations in mice and neurodegenerative disease in humans. At the cellular level, spectraplakins act as cross-linkers to physically bridge actin and microtubules. Despite these essential functions in the nervous system, we know very little about how spectraplakins are regulated at the molecular level. In this proposal, we will use Shot as a paradigm to ask how spectraplakins are regulated using cutting-edge single molecule visualization techniques. 1) We will establish an assay for visualizing Shot actin-microtubule cross-linking at single molecule resolution. 2) We will test the hypothesis that Shot is regulated by an intramolecular inhibition mechanism. Given the high degree of conservation among members of this family of proteins, we anticipate that these regulatory mechanisms will be conserved with vertebrate spectraplakins, as well.