Oscillatory Ca2 signaling in the C. elegans intestine

线虫肠道中的振荡 Ca2 信号传导

• 批准号: 7631168
• 负责人: KEVIN STRANGE
• 金额: $ 10.89万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-06-01 至 2009-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7631168
• 关键词: Apoptosis; Arrhythmia; Autoimmune Diseases; Behavior; Buffers; Caenorhabditis elegans; Calcium Oscillations; Calmodulin; Cations; Cell Culture Techniques; Cell Proliferation; Cell membrane; Cell physiology; Cells; Defecation; Diabetes Mellitus; Disease; Electrophysiology (science); Epithelial Cells; Event; Excision; Exhibits; Exocytosis; Feedback; Fertilization; Foundations; Frequencies; Gene Expression; Gene Transfer Techniques; Genes; Genetic; Genetic Epistasis; Genome; Heart Diseases; Homologous Gene; Image; Immunofluorescence Immunologic; Intestines; Knock-out; Laboratories; Malignant Neoplasms; Mediating; Membrane; Membrane Potentials; Methods; Molecular; Molecular Genetics; Muscle Contraction; Mutagenesis; Mutate; Nature; Nematoda; Physiological; Play; Potassium Channel; Preparation; Process; Property; Proteins; RNA Interference; Reagent; Regulation; Relative (related person); Research Personnel; Role; Signal Pathway; Signal Transduction; System; Testing; Time; cell motility; extracellular; genetic analysis; imaging modality; insight; intestinal epithelium; knock-down; knockout gene; mutant; novel; patch clamp; positional cloning; programs; receptor; sensor; tool

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.

描述（由申请人提供）：细胞质Ca2+水平控制着许多不同的细胞过程，包括基因表达，胞吐和分泌，运动性和收缩，细胞增殖，程序性细胞死亡和分化。尽管生理学家对CA2+信号事件有了令人印象深刻的了解，但许多基本问题仍未得到解决。线虫秀丽隐杆线虫为定义Ca2+信号传导的分子机制提供了许多实验优势。这些优点包括通过RNA干扰，敲除和转基因操纵基因表达的相对轻松和经济；现成的众多分子试剂和突变虫菌株的可用性；一个完全测序且宣布良好的基因组；以及执行诱变和正向遗传分析的能力。秀丽隐杆线虫中的后身体壁肌收缩（PBOC）驱动排便行为，每45-50秒以节奏方式发生。遗传分析已经确定了许多基因，这些基因在突变或敲击时会破坏PBOC节奏。这些包括编码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是由肠上皮中的节奏，依赖IPS依赖性细胞内Ca2+振荡驱动的。最近，我们开发了秀丽隐杆线虫细胞培养方法，该方法首次允许插图夹插座的肠细胞Ca2+电导。此外，我们开发了一种新型的孤立肠制剂，可允许细胞内CA2+振荡的生理表征。我们将使用Ca2+成像，电生理学，反向遗传学和免疫荧光的组合来检验PLC-P和PLC-Y，KCNQ通道KQT-2和KQT-3以及TRPM通道GON-2和GTL-1和GTL-1和GTL-1共同调节细胞内Ca2+释放。我们还将使用斑块夹电生理学和基因基因敲除来确定是否通过GON-2和/或GTL-1对TRPM样CA2+通道ORCA编码。我们将在我们的研究中使用的实验方法的组合更加昂贵和耗时，或者在脊椎动物实验系统中不可能。通过定义肠道Ca2+信号传导的基本方面，该提案构成了长期努力的基础，该基础将利用秀丽隐杆线虫的相当大的实验优势，以对不可启用的细胞振动CA2+信号通路产生综合的分子理解。鉴于CA2+信号传导的基本和高度保守的性质，从秀丽隐杆线虫获得的见解清楚地将提供对脊椎动物Ca2+信号传导机制的新的重要见解。对CA2+信号传导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众多疾病过程至关重要。