Novel subcellular and molecular tools to study Ca(2+) transport mechanisms during the elusive moulting stages of crustaceans: flow cytometry and polyclonal antibodies.

Abstract

Our understanding of calcium homeostasis during the crustacean moulting cycle derives from research on intermoult animals that has been extrapolated to other stages. In terms of transepithelial Ca(2+) flux, the more interesting stages are those surrounding ecdysis since crustaceans experience a sizeable negative calcium balance in immediate premoult and a significant positive calcium balance in immediate postmoult. These stages are elusive in the sense that larger species such as lobsters are rarely captured at this time, and smaller species such as blue crabs and crayfish are seldom synchronized in their moulting cycle. The reductionist approaches employed in cellular physiology, such as vesicle techniques, employ pooling of fresh tissues from many organisms. Examination of the elusive moulting stages requires more sensitive approaches that can utilize tissue from an individual crustacean to characterize Ca(2+) pumps (Sarco/Endoplasmic Reticulum Ca(2+)-ATPase, SERCA; Plasma Membrane Ca(2+)-ATPase, PMCA) and the Na(+)/Ca(2+) eXchanger (NCX). An emerging subcellular approach described in this paper is to use flow cytometry as a technique to monitor Ca(2+) uptake into Fluo-3-loaded membrane vesicles. This paper illustrates the utility of this technique for measuring ATP-dependent Ca(2+) uptake into hepatopancreatic basolateral membrane vesicles. Obstacles to progress in molecular studies have not been limited by synchronization of moulting since tissue can be snap-frozen and collected from many animals over time. Here, the problem has been the lack of specific antibodies that hybridize with the Ca(2+) transporters of interest so that they can be localized within epithelia. In this paper, we introduce polyclonal antibodies raised in rabbits against crayfish SERCA, PMCA and NCX. Immunocytochemistry of SERCA in muscle, PMCA in antennal gland and NCX in heart confirms the specificity of the antibodies.