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

描述（由申请人提供）：世界卫生组织估计，全世界有近5亿人患有各种形式的精神疾病，趋势数据预测，到2030年，抑郁症将成为世界上疾病负担的单一最高贡献者。许多精神健康障碍，包括抑郁症，与树突和突触接触的变化或破坏有关，树突和突触接触是由发育过程中控制形态的单个神经元内的动态过程建立的。神经元形态的一些最好的研究调节剂是Rho GTP酶，即Cdc 42（细胞分裂周期42）和Rac 1（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方面具有显著的相似性。以前的工作广泛使用这些蛋白质的野生型和突变形式的过表达，并导致一个流行的概念，即两者对细胞骨架动力学具有非常相似的影响。然而，有一个明显的功能分歧有待探讨。两者都是生理上必不可少的， 然而，它们在建立神经元形态方面的差异尚不清楚。Cdc 42和Rac 1的下游是一组效应物，它们似乎有助于神经突发育，被称为CRIB蛋白。确定Cdc 42和Rac 1何时何地被激活，以及它们激活的时间和位置如何影响它们在中枢神经元内的下游效应，对于理解神经元形态和突触连接的分子控制将是重要的。这些古老的信号机制是所有真核生物的基础，果蝇中可用的遗传工具将有助于他们的研究，同时也有助于深入了解哺乳动物的神经系统。我的中心假设是Cdc 42和Rac 1不是功能冗余的蛋白质，它们确实具有独特的信号传导特性，并且每个与下游CRIB效应子相互作用的时间和位置对于果蝇前角细胞（aCC）神经元的适当形态发育至关重要。本申请的目的是：（1）使用FRET aProbe构建体建立果蝇aCC神经元发育期间Rac 1的激活模式，补充先前在申办者实验室中证明的Cdc 42的激活模式;（2）使用比率和寿命FRET成像方法两者，观察Cdc 42和Rac 1在何处和何时与CRIB蛋白伴侣在体内结合，和（3）通过功能丧失研究确定CRIB蛋白伴侣如何促进神经元形态。通过确定Rho GTP酶的信号通路和它们对活神经元中神经元隔室形成的功能贡献，所提出的研究将提供关于神经元连接调节的重要信息。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会聚信号通路以生长神经突并形成突触。通过了解这些过程的分子基础，医学将获得新的治疗靶点，以对抗那些患有衰弱性神经心理疾病的病理生理学。

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.