Oscillatory Ca2 signaling in the C. elegans intestine

描述(申请人提供)：细胞质钙水平控制众多不同的细胞过程，包括基因表达、胞吐和分泌、运动和收缩、细胞增殖、细胞程序性死亡和分化。虽然生理学家已经对钙信号事件有了令人印象深刻的了解，但许多基本问题仍然没有答案。线虫线虫为确定钙信号的分子机制提供了许多实验优势。这些优势包括通过RNA干扰、敲除和转基因来操纵基因表达的相对容易和经济；大量分子试剂和突变蠕虫菌株的现成供应；完全测序和良好注释的基因组；以及执行突变和正向遗传分析的能力。线虫的后体壁肌肉收缩(PBOC)驱动排便行为，每45-50秒发生一次。基因分析已经确定了许多基因，当突变或被击倒时，会扰乱中国人民银行的节奏。这些基因包括编码IP3受体、PLC、K通道和TRPM阳离子通道的基因。生理和分子研究表明，PBOC是由肠上皮细胞内有节律性的、依赖于IPs的钙振荡所驱动的。最近，我们开发了原代线虫细胞培养方法，首次允许膜片钳表征肠道细胞的钙电导。此外，我们还开发了一种新的离体肠制剂，可以对细胞内钙振荡进行生理学表征。我们将结合钙成像、电生理学、反向遗传学和免疫荧光来验证PLC-p和PLC-y、KCNQ通道KQT-2和KQT-3以及TRPM通道Gon-2和GTL-1共同调节细胞内钙释放的假设。我们还将使用膜片钳电生理学和基因敲除来确定TRPM样钙通道orca是否由Gon-2和/或GTL-1编码。我们将在研究中使用的实验方法的组合大大增加了成本和时间，或者在脊椎动物实验系统中是不现实的。通过定义肠道钙信号的基本方面，这一建议形成了长期努力的重要基础，该努力将利用线虫的显著实验优势，发展对不可兴奋的细胞振荡钙信号通路的完整分子理解。鉴于钙信号的基本和高度保守的性质，从线虫中获得的见解显然将为脊椎动物的钙信号机制提供新的和重要的见解。对钙信号的详细分子理解对于理解和治疗包括癌症、心脏病和糖尿病在内的多种疾病过程是至关重要的。

DESCRIPTION (provided by applicant): Cytoplasmic Ca2+ levels control numerous, diverse cellular processes including gene expression, exocytosis and secretion, motility and contraction, cell proliferation, programmed cell death, and differentiation. While physiologists have gained an impressive understanding of Ca2+ signaling events, many fundamental questions remain unanswered. The nematode C. elegans provides numerous experimental advantages for defining molecular mechanisms of Ca2+ signaling. These advantages include relative ease and economy of manipulating gene expression by RNA interference, knockout and transgenesis; ready availability of numerous molecular reagents and mutant worm strains; a fully sequenced and well-annotated genome; and the ability to perform mutagenesis and forward genetic analysis. Posterior body wall muscle contraction (pBoc) in C. elegans drives defecation behavior and occurs in rhythmic fashion every 45-50 sec. Genetic analyses have identified numerous genes that, when mutated or knocked down, disrupt pBoc rhythm. These include genes encoding the IP3 receptor, PLC, K+ channels and TRPM cation channels. Physiological and molecular studies have demonstrated that pBoc is driven by rhythmic, IPs-dependent intracellular Ca2+ oscillations in the intestinal epithelium. Recently, we developed primary C. elegans cell culture methods that allow for the first time patch clamp characterization of intestinal cell Ca2+ conductances. In addition, we have developed a novel isolated intestine preparation that allows physiological characterization of intracellular Ca2+ oscillations. We will use a combination of Ca2+ imaging, electrophysiology, reverse genetics and immunofluorescence to test the hypothesis that PLC-p and PLC-y, the KCNQ channels KQT-2 and KQT-3, and the TRPM channels GON-2 and GTL-1 function together to regulate intracellular Ca2+ release. We will also use patch clamp electrophysiology and gene knockout to determine if the TRPM-like Ca2+ channel ORCa is encoded by gon-2 and/or gtl-1. The combination of experimental approaches we will use in our studies is substantially more costly and time-consuming, or not realistically possible in vertebrate experimental systems. By defining basic aspects of intestinal Ca2+ signaling, this proposal forms an essential foundation of a long-term effort that will exploit the considerable experimental advantages of C. elegans to develop an integrated molecular understanding of a non-excitable cell oscillatory Ca2+ signaling pathway. Given the fundamental and highly conserved nature of Ca2+ signaling, insights gained from C. elegans will clearly provide new and important insights into vertebrate Ca2+ signaling mechanisms. Detailed molecular understanding of Ca2+ signaling is essential for understanding and treating numerous disease processes including cancer, heart disease and diabetes.