Mitochondrial calcium uniporter in Drosophila transfers calcium between the endoplasmic reticulum and mitochondria in oxidative stress-induced cell death

Abstract

Mitochondrial calcium plays critical roles in diverse cellular processes ranging from energy metabolism to cell death. Previous studies have demonstrated that mitochondrial calcium uptake is mainly mediated by the mitochondrial calcium uniporter (MCU) complex. However, the roles of the MCU complex in calcium transport, signaling, and dysregulation by oxidative stress still remain unclear. Here, we confirmed that MCU contains evolutionarily conserved structures and requires essential MCU regulator (EMRE) for its calcium channel activities. We generated MCU loss-of-function mutants, which lacked mitochondrial calcium uptake in response to caffeine stimulation. Basal metabolic activities were not significantly affected in these MCU mutants, as observed in examinations of body weight, food intake, body sugar level, and starvation-induced autophagy. However, oxidative stress-induced increases in mitochondrial calcium, mitochondrial membrane potential depolarization, and cell death were prevented in these mutants. We also found that inositol 1,4,5-trisphosphate receptor genetically interacts with MCU and effectively modulates mitochondrial calcium uptake upon oxidative stress. Taken together, these results support the idea that MCU is responsible for endoplasmic reticulum-to-mitochondrial calcium transfer and for cell death due to mitochondrial dysfunction under oxidative stress.

A comprehensive genetic characterisation of the mitochondrial Ca2+ uniporter in Drosophila

“…The fact that MCU mutants are viable and fertile with no gross morphological or behavioural defects was initially surprising given the historical importance of mitochondrial Ca 2+ , but corroborates another recent report of fly MCU mutants [22], and is consistent with studies in mice and worms where deletion of the MCU orthologues are essentially benign at the organismal level under basal conditions [15,23]. Importantly, however, fly MCU mutants are significantly shorter-lived than controls.…”

“…Overexpression paradigms disrupting uniporter stoichiometry have previously been used to interrogate the functional relationships of uniporter components [22]. Using a classic eye morphology assay as a read-out of the impact of genetic interactions on cell and tissue viability, we first found that overexpression of any of the uniporter components alone in the eye, using a GMR-GAL4 driver, had no effect on eye or ommatidial morphology ( In contrast, co-expression of MCU with either MICU1 or MICU3 was not so detrimental, although in all cases it caused a very mild disruption of the ommatidial arrangement resulting in a mild 'roughened' appearance ( Fig.…”

“…The respiratory impairment could be due to the previously reported increase in oxidative stress that occurs in the MCU mutants [22], which has yet to be assessed in EMRE mutants. Alternatively, the short lifespan may be due to a myriad of potential metabolic imbalances, such as disruption of NADH/NAD+ levels.…”

Disarrangement of Endoplasmic reticulum-mitochondria communication impairs Ca2+homeostasis in FRDA

“…Our results with the overexpression of MCU are in favor of the hypothesis that increasing the Ca 2+ uptake by the mitochondria is neuroprotective in FRDA flies. Using different sensors to visualize and quantify mitochondrial Ca 2+ , overexpression of MCU has been shown to increase basal concentration of mitochondrial Ca 2+ as well as to enhance Ca 2+ uptake in drosophila cultured embryonic cells (Chaudhuri et al, 2016) and in living flies (Choi et al, 2017). Therefore, in our experimental scenario, overexpression of MCU would increase the mitochondrial Ca 2+ content and this would be sufficient to recover several loss-of frataxin phenotypes in the fly.…”

“…Furthermore, we have also observed that one of the consequences of reactivating MAMs in our cell culture model was the increase of mitochondrial Ca 2+ uptake. Therefore, we decided to genetically manipulate Ca 2+ transport into the mitochondria by altering the expression of the mitochondrial Ca 2+ uniporter (MCU), since misexpression of MCU or of other components of the MCU complex has been shown to change the mitochondrial Ca 2+ content (Choi et al, 2017;Drago & Davis, 2016;K.-S. Lee et al, 2018;Tufi et al, 2019). We observed that MCU mRNA levels were not altered in frataxin deficient flies ( Figure S3A).…”