Spatiotemporal Correlations between Cytosolic and Mitochondrial Ca2+ Signals Using a Novel Red-Shifted Mitochondrial Targeted Cameleon

Abstract

The transfer of Ca2+ from the cytosol into the lumen of mitochondria is a crucial process that impacts cell signaling in multiple ways. Cytosolic Ca2+ ([Ca2+]cyto) can be excellently quantified with the ratiometric Ca2+ probe fura-2, while genetically encoded Förster resonance energy transfer (FRET)-based fluorescent Ca2+ sensors, the cameleons, are efficiently used to specifically measure Ca2+ 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 [Ca2+]cyto and mitochondrial Ca2+ ([Ca2+]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 Ca2+ probe could be successfully targeted to mitochondria and was suitable to be used simultaneously with fura-2 to correlate [Ca2+]cyto and [Ca2+]mito within same individual cells. Our data indicate that depending on the kinetics of [Ca2+]cyto rises there is a significant lag between onset of [Ca2+]cyto and [Ca2+]mito signals, pointing to a certain threshold of [Ca2+]cyto necessary to activate mitochondrial Ca2+ uptake. The temporal correlation between [Ca2+]mito and [Ca2+]cyto as well as the efficiency of the transfer of Ca2+ from the cytosol into mitochondria varies between different cell types. Moreover, slow mitochondrial Ca2+ extrusion and a desensitization of mitochondrial Ca2+ uptake cause a clear difference in patterns of mitochondrial and cytosolic Ca2+ oscillations of pancreatic beta-cells in response to D-glucose.

Calcium signaling in pancreatic β-cells in health and in Type 2 diabetes

“…8G). Moreover, in line with another study [269], a threshold has to be reached in the cytosol before mitochondrial Ca 2+ uptake could begin (Fig. 8F) (Fig.…”

“…8A) [9,50,245,248,250,251,269], that occur simultaneously in individual ␤-cells within intact pancreatic islets [248]. They are paralleled but significantly delayed to oscillations in [Ca 2+ ] c ( Fig.…”

“…They are paralleled but significantly delayed to oscillations in [Ca 2+ ] c ( Fig. 8A-C) [9,50,245,269] [236,[270][271][272] remains to be properly addressed, although the silencing of MCU [9,242] and mitochondrial Ca 2+ buffering [273] do not seem to affect [Ca 2+ ] c changes while a silencing of NCLX reduced glucose-induced [Ca 2+ ] c increases [268].…”

The Pancreatic β-Cell: A Bioenergetic Perspective

“…However, it is unclear from the literature whether the apparent effects of elevated matrix Ca 2ϩ on insulin secretion can be ascribed purely to conventional bioenergetic effects, e.g., by enhancing ⌬ m , ⌬pH, or ⌬G p as a consequence of the activation of pyruvate, isocitrate, and 2-oxoglutarate dehydrogenases (510), whether mitochondrial Ca 2ϩ trans-port is required for the induction and maintenance of plasma membrane oscillations (13), or whether there are additional, less defined, effects relating to the generation of metabolic coupling factors (427, 537). (510,511,528).…”

Metabolism–Secretion Coupling and Mitochondrial Calcium Activities in Clonal Pancreatic β-Cells

“…However, the study of mitochondrial physiology in pancreatic b-cells is more or less limited to methods that allow the assessment of mitochondrial function in intact cells, since the quantity of b-cell mitochondria that can be isolated from primary islets is comparatively small and potentially contaminated with mitochondria from other islet cells. Yet, recent technical advancements in the field of fluorescent sensors have opened up new possibilities of studying mitochondrial function in one given cell on multiple levels in parallel (Tarasov, Griffiths, & Rutter, 2012;Waldeck-Weiermair et al, 2012). Such sensors can be used in primary cells, but are particularly applicable in b-cell lines in order to investigate basic concepts in b-cell physiology.…”