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

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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.