Genetic analysis of C. elegans cellular ormoregulation

线虫细胞或调节的遗传分析

• 批准号: 6754397
• 负责人: KEVIN STRANGE
• 金额: $ 15.1万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-01 至 2007-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6754397
• 关键词: Caenorhabditis elegans; alternatives to animals in research; biological signal transduction; biotechnology; cell biology; cell osmotic pressure; cell water; cytotoxicity; enzyme activity; functional /structural genomics; genetic manipulation; genetic models; genetic screening; helminth genetics; high performance liquid chromatography; homeostasis; kidney cell; kidney function; microarray technology; mitogen activated protein kinase; morphology; organic chemicals; osmotic pressure; solute; tissue /cell culture

DESCRIPTION (provided by applicant): The ability to tightly control solute and water balance during osmotic challenge is an essential prerequisite for cellular life. Osmotic homeostasis is maintained by regulated accumulation and loss of inorganic ions and organic osmolytes. Organic osmolytes play essential roles in protecting renal medullary cells from extreme hypertonic stress and fluctuating extracellular osmolality associated with the urinary concentrating mechanism. While cellular osmoregulation has been studied extensively in a variety of cell types, including kidney cells, major gaps exist in our molecular understanding of this essential process. The nematode C. elegans provides powerful experimental advantages for defining the genetic bases of fundamental biological processes. These advantages include a fully sequenced genome, genetic tractability, and ease and economy of manipulating gene function. Nematodes normally live in soil where environmental variables such as water availability and solute levels change constantly and dramatically. Recently, we demonstrated that C. elegans readily adapts to and survives extreme hypertonic stress. Given its many experimental advantages, C. elegans thus provides an outstanding model system in which to define the genes and genetic pathways responsible for cellular osmoregulation. This R21 grant application addresses stated objectives of the NIDDK PA entitled, "Pilot and feasibility program related to the kidney". We propose to characterize organic osmolyte homeostasis in C. elegans during adaptation to and recovery from hypertonic stress, perform whole genome microarray analyses to identify genes transcriptionally upregulated by hypertonicity, and assess the role of MAPK signaling pathways in cellular osmoregulation using mutant worm strains and RNAi. We will also perform mutagenesis screens to identify genes required for cellular osmoregulation in C. elegans. Powerful forward genetic screening methods have been used with great success to define molecular aspects of osmoregulation in bacteria and yeast, but have not been utilized previously to characterize this process in animal cells. Our proposed investigations represent a novel approach to the molecular study of animal cell osmotic homeostasis. Given the evolutionarily conserved nature of this process, studies in C. elegans will likely provide unique insights into osmoregulation, signaling mechanisms, and cellular stress and damage repair )rocesses in kidney cells as well as other mammalian cell types and tissues.

描述(由申请人提供)：在渗透挑战过程中严格控制溶质和水平衡的能力是细胞生命的基本先决条件。渗透平衡是通过调节无机离子和有机渗透调节物质的积累和损失来维持的。有机渗透调节剂在保护肾髓质细胞免受极端高渗应激和与尿液浓缩机制相关的波动的细胞外渗透压方面起着重要作用。虽然细胞渗透调节在包括肾细胞在内的各种细胞类型中得到了广泛的研究，但在我们对这一重要过程的分子理解上存在着重大的差距。 线虫为确定基本生物过程的遗传基础提供了强大的实验优势。这些优势包括完全测序的基因组，遗传易操纵性，以及操作基因功能的简单性和经济性。线虫通常生活在土壤中，那里的环境变量，如水的可获得性和溶质水平，不断和戏剧性地变化。最近，我们证明线虫很容易适应和生存极端的高渗胁迫。鉴于其许多实验优势，线虫因此提供了一个出色的模型系统，在其中定义负责细胞渗透调节的基因和遗传途径。 这份R21拨款申请针对的是NIDDK PA所述的目标，题为“与肾脏有关的试点和可行性计划”。我们建议表征线虫在适应和恢复高渗胁迫过程中的有机渗透压平衡，进行全基因组芯片分析以确定高渗上调的基因转录，并使用突变的蠕虫株和RNAi来评估MAPK信号通路在细胞渗透调节中的作用。我们还将进行突变筛选，以确定线虫细胞渗透调节所需的基因。强大的正向遗传筛选方法已经成功地用于确定细菌和酵母中渗透调节的分子方面，但以前还没有被用来在动物细胞中表征这一过程。我们提出的研究为动物细胞渗透平衡的分子研究提供了一种新的途径。鉴于这一过程在进化上的保守性，对线虫的研究可能会为了解肾脏细胞以及其他哺乳动物细胞和组织中的渗透调节、信号机制以及细胞应激和损伤修复过程提供独特的见解。