Rho GTPases and Synaptic Morphology: Visualizing the Proteome with FRET Imaging

Rho GTP 酶和突触形态：利用 FRET 成像可视化蛋白质组

基本信息

批准号：
8488316
负责人：
NIMA SHARIFAI
金额：
$ 4.72万
依托单位：
UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-06-01 至 2017-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8488316
关键词：
Amino Acid Sequence Anterior Anxiety Axon Binding Biochemical Bioprobe Botulinum Toxins Brain region Cell Cycle Cells Communication Complement Data Dendrites Development Disease Docking Double-Stranded RNA Drosophila genus Drosophila melanogaster Economic Burden Epidemic Eukaryota Event Fluorescence Fluorescence Resonance Energy Transfer Foundations Functional disorder Genetic Guanosine Triphosphate Phosphohydrolases Image Individual Kinetics Knock-out Laboratories Lead Left Life Link Location Mediating Medicine Membrane Mental Depression Mental disorders Molecular Morphology Nervous system structure Neurites Neurons Pattern Phenotype Physiology Process Property Protein Family Proteins Proteome Psyche structure RNA Interference Regulation Role Severities Signal Pathway Signal Transduction State Medicine Structure Synapses Time Transgenic Organisms Work World Health Organization axon growth base burden of illness cdc42 GTP-Binding Protein cognitive function combat effective therapy in vivo insight loss of function mutant neurite growth neuron development neuropsychological new therapeutic target preference protein expression ratiometric restoration rho GTP-Binding Proteins social symptomatic improvement synaptogenesis tool trend

项目摘要

DESCRIPTION (provided by applicant): The World Health Organization estimates that nearly 500 million people suffer from various forms of mental illness worldwide, and trending data predicts that by the year 2030 depression will be the single highest contributor to burden of disease in the world. Many mental health disorders, including depression, are associated with a change or disruption of dendrites and synaptic contacts, which are established by dynamic processes within individual neurons controlling morphology during development. Some of the best-studied regulators of neuron morphology are the Rho GTPases, namely Cdc42 (Cell division cycle 42) and Rac1 (Ras- related C3 botulinum toxin substrate). These proteins have a remarkable similarity in their amino acid sequence and apparent functional roles. Previous work extensively used over-expression of wild type and mutant forms of these proteins and lead to a popular notion that the two have very similar influences on cytoskeletal dynamics. However, there is an apparent functional divergence left to be explored. Both are physiologically essential, yet how they differ in establishing neuron morphology is unclear. Downstream to both Cdc42 and Rac1 is a group of effectors that appear to be instrumental in neurite development that are known as CRIB proteins. Determination of when and where Cdc42 and Rac1 are activated, and how the timing and location of their activation contribute to their downstream effects within central neurons will be important for understanding molecular control of neuronal morphology and synaptic connectivity. These ancient signaling mechanisms are fundamental to all eukaryotes, and genetic tools available in Drosophila melanogaster will facilitate their study while also giving insight into mammalian nervous systems. My central hypothesis is that Cdc42 and Rac1 are not functionally redundant proteins, that they indeed have unique signaling properties, and that timing and location of interactions each has with downstream CRIB effectors is crucial for proper morphological development in the Drosophila anterior corner cell (aCC) neuron. The objectives of this application are to: (1) establish the activation pattern of Rac1 during Drosophila aCC neuron development using a FRET aProbe construct, complementing that of Cdc42 demonstrated previously in the sponsor's laboratory; (2) see where and when Cdc42 and Rac1 bind with CRIB protein partners in vivo using both ratiometric and lifetime FRET imaging approaches, and (3) determine through loss of function studies how CRIB protein partners contribute to neuronal morphology. By determining for Rho GTPases both their signaling pathways and their functional contributions to the formation of neuronal compartments in living neurons, the proposed studies will provide important information on regulation of neuronal connections. A compelling aspect of this work will be visualizing how the neuron integrates converging signaling pathways to grow neurites and form synapses. By understanding the molecular basis of these processes, medicine will gain novel therapeutic targets to combat the pathophysiologies that afflict those with debilitating neuropsychological conditions.

描述（由申请人提供）：世界卫生组织估计，在全球范围内有近5亿人患有各种精神疾病，并且热门数据预测，到2030年，抑郁症将成为全球疾病负担的最高贡献者。许多精神健康障碍，包括抑郁症，都与树突和突触接触的变化或破坏有关，这些疾病是通过在发育过程中控制形态的单个神经元内动态过程确定的。神经元形态的一些最佳调节剂是Rho GTPases，即CDC42（细胞分裂周期42）和RAC1（RAS-相关的C3肉毒杆菌毒素底物）。这些蛋白质在其氨基酸序列和明显的功能作用上具有显着的相似性。以前的工作广泛地使用了这些蛋白质的野生型和突变形式的过表达，并导致一个流行的观念，即两者对细胞骨架动力学的影响非常相似。但是，还有一个明显的功能差异要探索。两者在生理上都是必不可少的，然而，它们在建立神经元形态方面的差异尚不清楚。 Cdc42和Rac1的下游是一组效应子，似乎在神经突发作中起了称为CRIB蛋白的发挥作用。确定何时何地激活Cdc42和Rac1，其激活的时间和位置如何促进中心神经元内的下游效应对于理解神经元形态和突触连通性的分子控制很重要。这些古老的信号传导机制对于所有真核生物都是基础的，果蝇中可用的遗传工具将促进他们的研究，同时也深入了解哺乳动物神经系统。我的中心假设是，cdc42和rac1在功能上不是冗余的蛋白质，它们确实具有独特的信号传导特性，并且每个与下游Crib效应子的相互作用的时机和相互作用的位置对于果蝇前角细胞（ACC）神经元的适当形态发展至关重要。该应用程序的目标是：（1）使用FRET APROBE构造在果蝇ACC神经元开发过程中建立Rac1的激活模式，并补充了先前在赞助商实验室中证明的CDC42；（2）请参阅Cdc42和Rac1的何处和何时使用比率和寿命FRET成像方法与Brib蛋白伴侣在体内结合，并且（3）通过功能丧失研究CRIB蛋白伴侣如何有助于神经元形态。通过确定其信号通路及其对活神经元中神经元区室形成的功能贡献的Rho GTPase，拟议的研究将提供有关调节神经元连接的重要信息。这项工作的一个引人注目的方面将是可视化神经元如何整合融合的信号通路以形成神经突并形成突触。通过了解这些过程的分子基础，医学将获得新颖的治疗靶标，以打击困扰那些具有令人衰弱的神经心理学状况的病理生理学。