Single molecule analysis of cytoskeletal cross-linking proteins

描述（申请人提供）：发育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作出反应，以建立具有适当目标的突触，并产生特定的轴突和树突结构。肌动蛋白丝和微管的细胞骨架网络作为支架，允许导航生长锥改变其轨迹，以响应吸引和排斥的引导线索，定义成熟神经元的形态，并在响应损伤时进行重塑。为了使所有这些过程正常进行，神经元必须以高度调节的方式协调肌动蛋白丝和微管的动态。本提案的目的是通过研究果蝇谱板短停（Shot）来了解神经元细胞骨架动力学是如何调节的。缺乏Shot的果蝇表现出轴突生长缺陷和中枢神经系统畸形。谱蛋白在动物谱系中是保守的，它们的突变导致小鼠感觉神经元变性和发育性脑畸形，以及人类神经退行性疾病。在细胞水平上，谱蛋白充当交联剂，在肌动蛋白和微管之间架起物理桥梁。尽管在神经系统中有这些重要的功能，但我们对光谱蛋白在分子水平上是如何调节的知之甚少。在本提案中，我们将使用Shot作为范例，询问如何使用尖端的单分子可视化技术调节光谱蛋白。1)我们将建立一种在单分子分辨率下可视化Shot actin-微管交联的方法。2)测试

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.