Single molecule analysis of cytoskeletal cross-linking proteins

描述（由申请人提供）：发育神经科学中的关键问题之一是理解不同类型的神经元如何响应外部信号以与其适当的靶点建立突触并产生其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一个支架，允许导航生长锥改变其轨迹，以响应吸引和排斥的指导线索，以定义成熟神经元的形态，并重塑损伤。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力学。本研究的目的是通过研究果蝇spectraplakin Short stop（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CNS畸形。Spectraplakins在动物谱系中是保守的，它们的突变导致小鼠的感觉神经元变性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，spectraplakins作为交联剂物理桥接肌动蛋白和微管。尽管在神经系统中有这些基本功能，但我们对spectraplakins在分子水平上的调控知之甚少。在这项提案中，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来调节spectraplakins。1)我们将建立一个测定可视化射击肌动蛋白微管交联在单分子分辨率。2)我们将测试 假设Shot受分子内抑制机制调节。鉴于该蛋白质家族成员之间的高度保守性，我们预计这些调节机制也将与脊椎动物spectraplakins一起保守。

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.