Spatiotemporal Correlations between Cytosolic and Mitochondrial Ca ²⁺ Signals Using a Novel Red-Shifted Mitochondrial Targeted Cameleon

Abstract

The transfer of Ca ²⁺ from the cytosol into the lumen of mitochondria is a crucial process that impacts cell signaling in multiple ways. Cytosolic Ca ²⁺ ([Ca ²⁺] _cyto) can be excellently quantified with the ratiometric Ca ²⁺ probe fura-2, while genetically encoded Förster resonance energy transfer (FRET)-based fluorescent Ca ²⁺ sensors, the cameleons, are efficiently used to specifically measure Ca ²⁺ within organelles. However, because of a significant overlap of the fura-2 emission with the spectra of the cyan and yellow fluorescent protein of most of the existing cameleons, the measurement of fura-2 and cameleons within one given cell is a complex task. In this study, we introduce a novel approach to simultaneously assess [Ca ²⁺] _cyto and mitochondrial Ca ²⁺ ([Ca ²⁺] _mito) signals at the single cell level. In order to eliminate the spectral overlap we developed a novel red-shifted cameleon, D1GO-Cam, in which the green and orange fluorescent proteins were used as the FRET pair. This ratiometric Ca ²⁺ probe could be successfully targeted to mitochondria and was suitable to be used simultaneously with fura-2 to correlate [Ca ²⁺] _cyto and [Ca ²⁺] _mito within same individual cells. Our data indicate that depending on the kinetics of [Ca ²⁺] _cyto rises there is a significant lag between onset of [Ca ²⁺] _cyto and [Ca ²⁺] _mito signals, pointing to a certain threshold of [Ca ²⁺] _cyto necessary to activate mitochondrial Ca ²⁺ uptake. The temporal correlation between [Ca ²⁺] _mito and [Ca ²⁺] _cyto as well as the efficiency of the transfer of Ca ²⁺ from the cytosol into mitochondria varies between different cell types. Moreover, slow mitochondrial Ca ²⁺ extrusion and a desensitization of mitochondrial Ca ²⁺ uptake cause a clear difference in patterns of mitochondrial and cytosolic Ca ²⁺ oscillations of pancreatic beta-cells in response to D-glucose.