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+ 水平控制许多不同的细胞过程，包括基因表达、胞吐作用和分泌、运动和收缩、细胞增殖、程序性细胞死亡和分化。虽然生理学家对 Ca2+ 信号传导事件有了深入的了解，但许多基本问题仍未得到解答。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、敲除和转基因操作基因表达相对容易和经济；大量分子试剂和突变蠕虫菌株的现成可用；完整测序且注释良好的基因组；以及进行诱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，并且每 45-50 秒有节奏地发生一次。遗传分析已发现许多基因，当突变或敲除时，会破坏 pBoc 节律。其中包括编码 IP3 受体、PLC、K+ 通道和 TRPM 阳离子通道的基因。生理学和分子研究表明，pBoc 是由肠上皮细胞内有节奏的、IPs 依赖性细胞内 Ca2+ 振荡驱动的。最近，我们开发了原代秀丽隐杆线虫细胞培养方法，首次实现了肠细胞 Ca2+ 电导的膜片钳表征。此外，我们还开发了一种新型离体肠道制剂，可以对细胞内 Ca2+ 振荡进行生理表征。我们将结合使用 Ca2+ 成像、电生理学、反向遗传学和免疫荧光来检验 PLC-p 和 PLC-y、KCNQ 通道 KQT-2 和 KQT-3 以及 TRPM 通道 GON-2 和 GTL-1 的假设共同调节细胞内 Ca2+ 的释放。我们还将使用膜片钳电生理学和基因敲除来确定 TRPM 样 Ca2+ 通道 ORCa 是否由 gon-2 和/或 gtl-1 编码。我们在研究中使用的实验方法的组合要昂贵得多且耗时，或者在脊椎动物实验系统中实际上是不可能的。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重要基础，该长期努力将利用秀丽隐杆线虫的大量实验优势来发展对非兴奋性细胞振荡 Ca2+ 信号传导途径的综合分子理解。鉴于 Ca2+ 信号传导的基本和高度保守的性质，从秀丽隐杆线虫中获得的见解显然将为脊椎动物 Ca2+ 信号传导机制提供新的重要见解。对 Ca2+ 信号传导的详细分子了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至关重要。