Differential Gel Electrophoresis and Transgenic Mitochondrial Calcium Reporters Demonstrate Spatiotemporal Filtering in Calcium Control of Mitochondria

Abstract

Mitochondria must adjust both their intracellular location and their metabolism in order to balance their output to the needs of the cell. Here we show by the proteomic technique of time series difference gel electrophoresis that a major result of neuroendocrine stimulation of the Drosophila renal tubule is an extensive remodeling of the mitochondrial matrix. By generating Drosophila that were transgenic for both luminescent and fluorescent mitochondrial calcium reporters, it was shown that mitochondrial calcium tracked the slow (minutes) but not the rapid (<1 s) changes in cytoplasmic calcium and that this resulted in both increased mitochondrial membrane polarization and elevated cellular ATP levels. The selective V-ATPase inhibitor, bafilomycin, further enhanced ATP levels, suggesting that the apical plasma membrane V-ATPase is a major consumer of ATP. Both the mitochondrial calcium signal and the increase in ATP were abolished by the mitochondrial calcium uniporter blocker Ru360. By using both mitochondrial calcium imaging and the potential sensing dye JC-1, the apical mitochondria of principal cells were found to be selectively responsive to neuropeptide signaling. As the ultimate target is the V-ATPase in the apical plasma membrane, this selective activation of mitochondria is clearly adaptive. The results highlight the dynamic nature and both spatial and temporal heterogeneity of calcium signaling possible in differentiated, organotypic cells and provide a new model for neuroendocrine control of V-ATPase.