In vitro and in vivo calibration of low affinity genetic Ca2+ indicators.

Calcium is a universal intracellular messenger and proper Ca2+concentrations ([Ca2+]) both in the cytosol and in the lumen of cytoplasmic organelles are essential for cell functions. Ca2+ homeostasis is achieved by a delicate pump/leak balance both at the plasma membrane and at the endomembranes, and improper Ca2+ levels result in malfunction and disease. Selective intraorganellar Ca2+measurements are best achieved by using targeted genetically encoded Ca2+ indicators (GECIs) but to calibrate the luminal fluorescent signals into accurate [Ca2+] is challenging, especially in vivo, due to the difficulty to normalize and calibrate the fluorescent signal in various tissues or conditions. We report here a procedure to calibrate the ratiometric signal of GAP (GFP-Aequorin Protein) targeted to the endo-sarcoplasmic reticulum (ER/SR) into [Ca2+]ER/SR based on imaging of fluorescence after heating the tissue at 50-52 °C, since this value coincides with that obtained in the absence of Ca2+ (Rmin). Knowledge of the dynamic range (Rmax/Rmin) and the Ca2+-affinity (KD) of the indicator permits calculation of [Ca2+] by applying a simple algorithm. We have validated this procedure in vitro using several cell types (HeLa, HEK 293T and mouse astrocytes), as well as in vivo in Drosophila. Moreover, this methodology is applicable to other low Ca2+ affinity green and red GECIs.

Sarcoplasmic reticulum Ca2+ decreases with age and correlates with the decline in muscle function in Drosophila.

Sarcopenia, the loss of muscle mass and strength associated with age, has been linked to impairment of the cytosolic Ca2+ peak that triggers muscle contraction, but mechanistic details remain unknown. Here we explore the hypothesis that a reduction in sarcoplasmic reticulum (SR) Ca2+ concentration ([Ca2+]SR) is at the origin of this loss of Ca2+ homeostasis. We engineered Drosophila melanogaster to express the Ca2+ indicator GAP3 targeted to muscle SR, and we developed a new method to calibrate the signal into [Ca2+]SRin vivo [Ca2+]SR fell with age from ∼600 µM to 50 µM in close correlation with muscle function, which declined monotonically when [Ca2+]SR was <400 µM. [Ca2+]SR results from the pump-leak steady state at the SR membrane. However, changes in expression of the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pump and of the ryanodine receptor leak were too modest to explain the large changes seen in [Ca2+]SR Instead, these changes are compatible with increased leakiness through the ryanodine receptor as the main determinant of the [Ca2+]SR decline in aging muscle. In contrast, there were no changes in endoplasmic reticulum [Ca2+] with age in brain neurons.This article has an associated First Person interview with the first author of the paper.

Caffeine chelates calcium in the lumen of the endoplasmic reticulum.

Cytosolic Ca2+ signals are often amplified by massive calcium release from the endoplasmic reticulum (ER). This calcium-induced calcium release (CICR) occurs by activation of an ER Ca2+ channel, the ryanodine receptor (RyR), which is facilitated by both cytosolic- and ER Ca2+ levels. Caffeine sensitizes RyR to Ca2+ and promotes ER Ca2+ release at basal cytosolic Ca2+ levels. This outcome is frequently used as a readout for the presence of CICR. By monitoring ER luminal Ca2+ with the low-affinity genetic Ca2+ probe erGAP3, we find here that application of 50 mM caffeine rapidly reduces the Ca2+ content of the ER in HeLa cells by ∼50%. Interestingly, this apparent ER Ca2+ release does not go along with the expected cytosolic Ca2+ increase. These results can be explained by Ca2+ chelation by caffeine inside the ER. Ca2+-overloaded mitochondria also display a drop of the matrix Ca2+ concentration upon caffeine addition. In contrast, in the cytosol, with a low free Ca2+ concentration (10-7 M), no chelation is observed. Expression of RyR3 sensitizes the responses to caffeine with effects both in the ER (increase in Ca2+ release) and in the cytosol (increase in Ca2+ peak) at low caffeine concentrations (0.3-1 mM) that have no effects in control cells. Our results illustrate the fact that simultaneous monitoring of both cytosolic- and ER Ca2+ are necessary to understand the action of caffeine and raise concerns against the use of high concentrations of caffeine as a readout of the presence of CICR.

Systematic Identification of MCU Modulators by Orthogonal Interspecies Chemical Screening.

The mitochondrial calcium uniporter complex is essential for calcium (Ca2+) uptake into mitochondria of all mammalian tissues, where it regulates bioenergetics, cell death, and Ca2+ signal transduction. Despite its involvement in several human diseases, we currently lack pharmacological agents for targeting uniporter activity. Here we introduce a high-throughput assay that selects for human MCU-specific small-molecule modulators in primary drug screens. Using isolated yeast mitochondria, reconstituted with human MCU, its essential regulator EMRE, and aequorin, and exploiting a D-lactate- and mannitol/sucrose-based bioenergetic shunt that greatly minimizes false-positive hits, we identify mitoxantrone out of more than 600 clinically approved drugs as a direct selective inhibitor of human MCU. We validate mitoxantrone in orthogonal mammalian cell-based assays, demonstrating that our screening approach is an effective and robust tool for MCU-specific drug discovery and, more generally, for the identification of compounds that target mitochondrial functions.