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

这项建议的目的是利用小龙虾蜕皮模型来表征外膜和内膜上的钙泵和钙交换器(钠/钙交换器)，编码它们的基因，以及可能调节这些基因的类固醇激素。蜕皮后为上皮钙转运蛋白的上调/激活提供了一个自然的模型，因为小龙虾从蜕皮间的钙平衡过渡到令人印象深刻的跨初级交换上皮的单向钙内流(2 mmol/kg/h)。细胞和亚细胞内的钙同源酶在大量的钙跨上皮运输过程中都受到挑战。需要检验的假设是，一系列钙转运蛋白共同作用，在小龙虾细胞中实现钙稳态。在蜕皮后(实验)，与蜕皮间(基线水平，对照)相比，增强的跨皮钙单向内流与这些蛋白的活性或表达的协调变化有关。被研究的小龙虾组织既有上皮细胞，也有非上皮细胞(肌肉)。与蜕皮间(钙平衡，对照)不同，蜕皮后钙泵和NCX的特征是(实验中的跨皮钙内流)。1.体外研究钙转运蛋白的生理特性：通过流式细胞术检测钙敏感染料Fluo-3的平均荧光强度(与侧向散射)的变化，研究基底外侧膜囊泡(BLMV)摄取依赖于ATP和Na的钙离子的动力学和药理学。这项技术将通过将抗体与细胞内表位结合来进一步完善，使内向外的囊泡能够被分类。同时，在BLMV和SR/ER制备的微粒体中将测定放射性标记的钙的快速过滤摄取，以分别确定外膜和内膜上的钙转运体的动力学/药理学。2.纯化的钙转运蛋白的分子特征及其进化：将利用标准分子技术克隆小龙虾PMCA和NCX的全长cDNA。构建这些古老基因家族的系统发育树将被用来估计它们的进化速度。3.钙转运蛋白编码基因的表达调控：将使用标准分子技术对钙转运蛋白的表达进行量化。免疫细胞化学纯化抗体定位钙转运蛋白：已成功地制备了针对小龙虾SERCA/PMCA/NCX的抗体。免疫细胞化学定位转运蛋白的组织分布(采用明场、荧光)和亚细胞分布(激光扫描共聚焦、电子显微镜)。钙转运体编码基因的调节：编码钙转运体的基因的调节将通过基因组DNA的特征来确定。钙稳态具有深刻的生物学意义。在多个上皮细胞上整合相关的钙转运蛋白及其基因的变化，将从有机体的角度描绘出钙稳态。该项目还将加强对代表性不足群体的学生的研究培训。

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.