G Protein Signaling in the C. elegans Nervous System

描述（由申请人提供）：我们试图了解神经递质如何通过异源三聚体G蛋白信号来调节神经元的活动。成瘾药物激活G蛋白偶联受体，许多精神疾病是由于G蛋白的神经传递改变，因此了解神经元中G蛋白信号传导的基本机制至关重要。Galpha-o是由G蛋白偶联的神经递质受体激活的主要脑G蛋白，但其信号传导机制尚不清楚。最近的一项发现是，在细胞分裂过程中，alpha-o调节微管上的力，这提出了一种假设，即alpha-o也可能通过微管力在神经元中起作用，改变细胞结构。gal -o信号抑制线虫的产卵行为，我们的第一个目标是以此为模型来研究gal -o信号的机制。因此，我们将鉴定产卵系统中产生和接收α -o介导信号的细胞。我们将分析三个新的基因，我们已经从遗传学上确定了这一信号所需的基因，其中一个我们已经克隆并显示编码TRP离子通道。我们将通过荧光标记野生型和α - 0突变动物的产卵系统的神经过程和突触，来验证α - 0通过细胞骨架改变神经元结构的假设。我们的第二个目标是利用alpha-o信号的第二个模型。因此，我们将研究由5 -羟色胺介导的信号传导机制，5 -羟色胺是一种参与人类抑郁症的神经递质。我们正在筛选对血清素没有反应的秀丽隐杆线虫的突变体。我们将完成这个筛选，克隆和分析一个新的血清素信号基因，我们已经通过这种方法确定。我们还将分析一组秀丽隐杆线虫血清素受体同源物的表达模式和敲除表型。我们的第三个目标是识别和分析在alpha-o下游发挥作用以调节其作用的分子。我们将完成一项基因筛选，找出干扰α -o下游神经递质信号的突变体。利用这种筛选和血清素筛选，我们已经分离出五种突变，它们似乎既破坏了α -o介导的神经传递，也破坏了分裂细胞中微管力的产生。这有力地支持了一种假设，即alpha-o在这两种功能中通过相同的机制起作用。通过上位分析，其中三个突变确定了一个单一基因，该基因似乎在alpha-o的下游起作用。我们将克隆并分析由这些突变确定的基因。

DESCRIPTION (provided by applicant): We seek to understand how neurotransmitters signal through heterotrimeric G proteins to modulate the activities of neurons. Addictive drugs activate G protein-coupled receptors, and a number of mental diseases are due to alterations in neurotransmission through G proteins, so it is critical to understand the fundamental mechanisms of G protein signaling in neurons. Galpha-o is the major brain G protein activated by G protein-coupled neurotransmitter receptors, but little is known about its mechanism of signaling. A recent discovery is that Galpha-o acts in dividing cells to regulate force on microtubules, raising the hypothesis that Galpha-o may also act in neurons via microtubule force to modify cell structure. Galpha-o signaling inhibits egg-laying behavior in C. elegans, and our first aim is to use this as a model to study the Galpha-o signaling mechanism. Thus we will identify the cells of the egg-laying system that generate and receive Galpha-o-mediated signal(s). We will analyze three new genes we have genetically identified that are required for this signaling, one of which we have already cloned and shown to encode a TRP ion channel. We will test the hypothesis that Galpha-o acts via the cytoskeleton to alter the structure of neurons by fluorescently labeling the neural processes and synapses of the egg-laying system in wild-type and Galpha-o-mutant animals. Our second aim exploits a second model for Galpha-o signaling. Thus we will study the mechanism of Galpha-o-mediated signaling by serotonin, a neurotransmitter involved in depression in humans. We are screening for mutants of C. elegans that fail to respond to serotonin. We will complete this screen and clone and analyze a new serotonin signaling gene that we have already identified by this approach. We will also analyze the expression patterns and knockout phenotypes for a set of C. elegans serotonin receptor homologs. Our third aim is to identify and analyze the molecules that act downstream of Galpha-o to mediate its effects. We will complete a genetic screen for mutants that disrupt neurotransmitter signaling downstream of Galpha-o. Using this screen and the serotonin screen, we have already isolated five mutations that appear to disrupt both Galpha-o-mediated neurotransmission as well as microtubule force generation in dividing cells. This strongly supports the hypothesis that Galpha-o acts by the same mechanism for both functions. Three of the mutations identify a single gene that, by epistasis analysis, appears to function downstream of Galpha-o. We will clone and analyze the gene(s) identified by these mutations.