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

本研究的目的是利用小龙虾的换壳模型来表征小龙虾内外膜上的Ca2+泵和Ca2+交换器（Na+/Ca2+交换器），编码它们的基因，以及推测调节这些基因的类固醇激素。当小龙虾从脱毛间Ca2+平衡过渡到令人印象深刻的单向Ca2+内流（2 mmol/kg/h）时，脱毛后为上皮Ca2+转运蛋白的上调/激活提供了一个自然模型。细胞和亚细胞Ca2+稳态在大量Ca2+在上皮运输过程中受到挑战。要测试的假设是，一套Ca2+运输蛋白一起工作，以实现Ca2+在小龙虾细胞内的稳态。在脱毛后（实验），与脱毛期间（基线水平，对照）相比，增强的上皮内Ca2+单向内流与这些蛋白的活性或表达的协调变化有关。所研究的小龙虾组织既有上皮细胞，也有非上皮细胞（肌肉）。Ca2+泵和NCX将在脱毛后（经上皮Ca2+内流，实验）被表征，而不是在脱毛期间（Ca2+平衡，控制）。具体目标是：1。通过体外技术Ca2+转运体的生理表征：ATP和Na+依赖性Ca2+摄取到基底外侧膜泡（BLMV）的动力学和药理学将使用流式细胞术来检测Ca2+敏感染料fluo-3的平均荧光强度（相对于侧散射）的变化。这项技术将通过结合细胞内表位的抗体进一步完善，使内外囊泡能够被分类。同时，将放射性标记Ca2+的快速过滤摄取测定到BLMV以及SR/ER制备的微粒体中，分别测定Ca2+转运体在膜外或膜内的动力学/药理学。2. 纯化的Ca2+转运蛋白的分子特征及其进化：利用标准分子技术克隆小龙虾PMCA和NCX的完整cDNA。这些古老基因家族的系统发育树的构建将用于估计它们的进化速度。3. 编码Ca2+转运蛋白的基因表达调控：Ca2+转运蛋白的表达将使用标准分子技术进行量化。使用纯化抗体定位Ca2+转运体用于免疫细胞化学：抗体已经成功地针对小龙虾SERCA/PMCA/NCX。免疫细胞化学将用于定位转运体的组织分布（使用亮场，荧光）和它们的亚细胞分布（激光扫描共聚焦，电子显微镜）。编码Ca2+转运蛋白的基因调控：编码Ca2+转运蛋白的基因调控将通过基因组DNA的特征来确定。Ca2+稳态具有深远的生物学意义。整合Ca2+转运蛋白及其基因在多个上皮细胞中的相关变化将从机体环境中描述Ca2+稳态。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.