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

描述(申请人提供)：世界卫生组织估计，全球有近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最大的单一因素。许多精神健康障碍，包括抑郁症，都与树突和突触联系的变化或中断有关，这些联系是由个体神经元内控制发育过程中的形态的动态过程建立的。一些研究最深入的神经元形态调节因子是Rho GTP酶，即CDC42(细胞分裂周期42)和rac1(RAS相关的C3肉毒毒素底物)。这些蛋白质在氨基酸序列和明显的功能作用上具有显著的相似性。以前的工作广泛使用了这些蛋白的野生型和突变型的过度表达，并导致了一种流行的观点，即这两种蛋白对细胞骨架动力学的影响非常相似。然而，还有一个明显的功能分歧有待探索。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不同之处尚不清楚。在cdc42和rac1的下游都有一组效应器，它们似乎对轴突的发育起到了重要作用，它们被称为婴儿床蛋白。确定CDC42和rac1被激活的时间和地点，以及它们被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，而在果蝇身上可用的遗传工具将有助于它们的研究，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，CDC42和rac1在功能上不是多余的蛋白质，它们确实具有独特的信号特性，并且每个蛋白质与下游CRIBB效应器相互作用的时间和位置对于果蝇前角细胞(ACC)神经元的正常形态发育至关重要。本申请的目的是：(1)使用FRET aProbe构建方法建立果蝇ACC神经元发育过程中rac1的激活模式，补充先前在赞助者实验室中展示的CDc42的激活模式；(2)使用比率计量学和终身FRET成像方法，了解CDC42和rac1在体内何时何地与CRIB蛋白伙伴结合；以及(3)通过功能丧失研究确定CRIB蛋白伙伴如何对神经元形态做出贡献。通过确定Rho GTP酶的信号通路和它们在活神经元中形成神经室的功能贡献，拟议的研究将为神经元连接的调控提供重要信息。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汇聚的信号通路来生长轴突和形成突触。通过了解这些过程的分子基础，医学将获得新的治疗靶点，以对抗困扰那些患有衰弱神经心理疾病的人的病理生理学。

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.