RUI: Calcium Homeostasis Modeled on the Freshwater Crayfish Molting Cycle: From Physiology to Molecular Regulation

本提案的目标是使用小龙虾蜕皮模型来表征外膜和内膜上的Ca 2+泵和Ca 2+交换器（Na+/Ca 2+交换器）、编码它们的基因以及puronectin调节基因的类固醇激素。 蜕皮后提供了一个天然的模型上调/激活上皮细胞Ca 2+转运蛋白的小龙虾过渡从蜕皮间Ca 2+平衡令人印象深刻的单向Ca 2+流入（2 mmol/kg/h）整个初级交换上皮。 细胞和亚细胞Ca 2+稳态在大量Ca 2+通过上皮细胞期间受到挑战。 有待检验的假设是，一套钙转运蛋白共同努力，以实现Ca 2+稳态的小龙虾细胞。 在蜕皮后（实验），增强transepithelial单向流入的Ca 2+与协调的变化，这些蛋白质的活性或表达相比，蜕皮（基线水平，控制）。待研究的小龙虾组织是上皮细胞以及非上皮细胞（肌肉）。 将在蜕皮后（经上皮Ca 2+内流，实验）与蜕皮间（Ca 2+平衡，对照）期间表征Ca 2+泵和NCX。 具体目标是：1.通过体外技术对Ca 2+转运蛋白进行生理学表征：将使用流式细胞术研究ATP和Na+依赖性Ca 2+摄取至基底外侧膜囊泡（BLMV）的动力学和药理学，以检测Ca 2+敏感染料fluo-3的平均荧光强度（相对于侧向散射）的变化。 该技术将通过将抗体结合到细胞内表位来进一步完善，从而使由内而外的囊泡能够被分选。 同时，将测定BLMV以及SR/ER制备的微粒体对放射性标记Ca 2+的快速过滤摄取，以分别测定外膜或内膜上Ca 2+转运蛋白的动力学/药理学。 2.纯化的Ca 2+转运蛋白的分子表征及其进化：将使用标准分子技术克隆小龙虾PMCA和NCX的完整cDNA。 这些古老的基因家族的系统发育树的构建将被用来估计他们的进化速度。3.编码Ca 2+转运蛋白的基因表达的调节：将使用标准分子技术定量Ca 2+转运蛋白的表达。使用纯化的抗体进行免疫细胞化学的Ca 2+转运蛋白的定位：已经成功地针对小龙虾SERCA/PMCA/NCX产生了抗体。免疫细胞化学将用于定位转运蛋白的组织分布（使用明视野、落射荧光）和它们的亚细胞分布（激光扫描共聚焦、电子显微镜）. Ca 2+转运蛋白基因的调控：Ca 2+转运蛋白基因的调控将通过对基因组DNA的分析来确定。 整合相关的变化，在Ca 2+转运蛋白和它们的基因在多个上皮细胞将描绘Ca 2+稳态从器官的背景。 该项目还将加强对代表性不足群体学生的研究培训。

The goal of this proposal is to use the crayfish molting model to characterize the Ca2+ pump and Ca2+ exchanger (Na+/Ca2+ exchanger) on external and internal membranes, the genes that encode them, and the steroid hormone that putatively regulates the genes. Postmolt provides a natural model for upregulation/activation of epithelial Ca2+ transporters as crayfish transition from intermolt Ca2+ balance to impressive unidirectional Ca2+ influx (2 mmol/kg/h) across the primary exchange epithelia. Both cellular and subcellular Ca2+ homeostases are challenged during mass Ca2+ transit across epithelia. The hypothesis to be tested is that a suite of Ca2+ transporting proteins work together to achieve Ca2+ homeostasis in crayfish cells. During postmolt (experimental), enhanced transepithelial unidirectional influx of Ca2+ is associated with coordinated changes in activity or expression of these proteins compared with intermolt (baseline levels, control). Crayfish tissues to be studied are epithelia as well as non-epithelial cells (muscle). The Ca2+ pump and NCX will be characterized during postmolt (transepithelial Ca2+ influx, experimental) as opposed to intermolt (Ca2+ balance, control). The specific aims are:1. Physiological characterization of Ca2+ transporters through in vitro techniques: The kinetics and pharmacology of ATP- and Na+-dependent Ca2+ uptake into basolateral membrane vesicles (BLMV) will be studied using flow cytometry to detect change in mean fluorescence intensity (versus side scatter) of the Ca2+ sensitive dye fluo-3. The technique will be further refined through binding antibodies to intracellular epitopes, enabling inside out vesicles to be sorted. At the same time, rapid filtration uptake of radiolabelled Ca2+ will be determined into BLMV as well as microsomes prepared from SR/ER to determine the kinetics/pharmacology of Ca2+ transporters on external or internal membranes respectively. 2. Molecular characterization of purified Ca2+ transporting proteins and their evolution: The complete cDNA of crayfish PMCA and NCX will be cloned using standard molecular techniques. Construction of phylogenetic trees of these ancient gene families will be used to estimate their rates of evolution. 3. Regulation of expression of genes encoding Ca2+ transporting proteins: Expression of Ca2+ transporters will be quantified using standard molecular techniques.4. Localization of Ca2+ transporters using purified antibodies for immunocytochemistry: Antibodies have been successfully raised against crayfish SERCA/PMCA/NCX. Immunocytochemistry will be used to locate the tissue distribution of the transporters (using bright field, epifluorescence) and their subcellular distribution (laser scanning confocal, electron microscopy).5. Regulation of genes encoding Ca2+ transporters: Regulation of genes encoding the Ca2+ transporters will be determined through characterizing genomic DNA.Ca2+ homeostasis has profound biological relevance. Integrating the associated changes in Ca2+ transporting proteins and their genes at multiple epithelia will delineate Ca2+ homeostasis from an organismic context. The project will also enhance research training of students from underrepresented groups.