Genetic analysis of C. elegans cellular ormoregulation

描述（由申请方提供）：渗透压挑战期间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物的积累和损失来维持的。有机渗透压调节剂在保护肾髓质细胞免受与尿浓缩机制相关的极端高渗应激和细胞外渗透压波动中起重要作用。虽然细胞凋亡调控已经在包括肾细胞在内的各种细胞类型中进行了广泛的研究，但我们对这一重要过程的分子理解存在重大差距。 线虫C.秀丽隐杆线虫为确定基本生物过程的遗传基础提供了强有力的实验优势。这些优势包括全测序的基因组、遗传易处理性以及操纵基因功能的简便和经济。线虫通常生活在土壤中，土壤中的环境变量，如水的可用性和溶质水平不断变化和显着。最近，我们证明了C。秀丽线虫很容易适应极端的高渗压力并存活下来。鉴于它的许多实验优势，C。因此，秀丽线虫提供了一个杰出的模型系统，在其中定义了负责细胞凋亡调节的基因和遗传途径。 该R21赠款申请解决了NIDDK PA的既定目标，标题为“与肾脏相关的试点和可行性计划”。我们建议在C.线虫在适应高渗应激和从高渗应激中恢复的过程中，进行全基因组微阵列分析以鉴定由高渗性转录上调的基因，并使用突变蠕虫菌株和RNAi评估MAPK信号传导途径在细胞凋亡调节中的作用。我们还将进行诱变筛选，以确定在C.优雅强大的正向遗传筛选方法已被用于确定细菌和酵母中的α-淀粉酶调节的分子方面，取得了巨大的成功，但以前还没有被用于表征动物细胞中的这一过程。我们提出的调查代表了一种新的方法，动物细胞渗透稳态的分子研究。考虑到这一过程在进化上的保守性，C. elegans将可能为肾细胞以及其他哺乳动物细胞类型和组织中的信号调节、信号机制和细胞应激和损伤修复过程提供独特的见解。

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.