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

Abstract

Mitochondrial Ca 2+ uptake is an important mediator of metabolism and cell death. Identification of components of the highly conserved mitochondrial Ca 2+ uniporter has opened it up to genetic analysis in model organisms. Here we report a comprehensive genetic characterisation of the known uniporter components conserved in Drosophila. While loss of MCU or EMRE abolishes fast mitochondrial Ca 2+ uptake, this results in surprisingly mild phenotypes. In contrast, loss of the regulatory gatekeeper component MICU1 has a much more severe phenotype, being developmental lethal, consistent with unregulated Ca 2+ uptake. Mutants for MICU3 are viable with mild neurological phenotypes. Genetic interaction studies reveal that MICU1 and MICU3 are not functionally interchangeable. More surprisingly, loss of MCU or EMRE does not suppress MICU1 mutant lethality, suggesting that the lethality results from MCU-independent functions. This study helps shed light on the physiological requirements of the mitochondrial Ca 2+ uniporter, and provides a suite of tools to interrogate their interplay in homeostasis and disease conditions. B. DNA analysis of MCU 1 . Primer sequences are detailed in the 'Materials and Methods' section.The control genotype yielded a ~2.5 kb band, compared to ~900 bp for MCU 1 homozygotes. C. Western blot analysis of MCU 1 . Immunoblots were probed with the indicated antibodies. Asterisk denotes a non-specific band. Mitochondrial ATP5A is used as a loading control. D.Representative traces of Ca 2+ uptake in mitochondria isolated from adult flies of the indicated genotypes after addition of 45 μM CaCl2. Extramitochondrial Ca 2+ was measured by Calcium Green-5N fluorescence. Ca 2+ was released from mitochondria by addition of 1 μM FCCP. a.u.: arbitrary units. The control genotype is w 1118 . Addition of the MCU inhibitor Ruthenium Red (RuR; 2 μM) blocks mitochondrial Ca 2+ uptake, which is mirrored by MCU 1 . Mitochondrial Ca 2+ uptake is restored by transgenic re-expression of MCU. E. Relative uptake kinetics were determined through linear fits of the initial phase of Ca 2+ uptake and normalized to the wild type control (mean ± SEM, n = 3). Figure 2. MCU 1 shortens lifespan without impacting organismal phenotypes despite respiratory defects. A. The percentage of adult flies eclosing as homozygous MCU 1 mutants versus balanced heterozygotes, together with the expected Mendelian ratio in the offspring (n > 700). B. Lifespan curves of MCU 1 male flies compared with control (w 1118 ) and transgenic rescue (MCU 1 + MCU). Statistical analysis: Mantel-Cox log-rank test (n ≧ 74). C. Climbing assay of control (da/+) and MCU 1 flies, 2 and 20 days post-eclosion. Significance measured by Kruskal-Wallis test with Dunn's post-hoc correction for multiple comparisons (mean ± 95% confidence interval; n > 50; ns, non-significant). D. Relative ATP levels from control and MCU 1 flies. Statistical analysis: unpaired t-test (mean ± SD; n 2-3; **** P < 0.0001, ns, non-significant). E, F. Oxygen consumption rate (OCR) of control and MC...

SMDT1variants impair EMRE-mediated mitochondrial calcium uptake in patients with muscle involvement

“…5G). These results are compatible with other studies demonstrating that: (1) Smdt1 -/flies or mice exhibit normal routine mitochondrial respiration (42,43) In this way, increased mitochondrial Ca 2+ uptake might be responsible for the increased OCR and ECAR in non-stimulated P3-MICU1 cells, compatible with a higher metabolic activity (Fig. 5H).…”

“…4D), supporting the above conclusion that the absence of EMRE protein is causally linked to MCUsubcomplex formation and impairment of MCU-mediated mitochondrial Ca 2+ uptake. The latter was not due to Dy depolarization, compatible with other EMRE deficiency models (28,42,67) 1), normal routine OCR (Fig. 5E), normal ATP-linked OCR (Fig.…”

Inactivation of the mitochondrial protease Afg3l2 results in severely diminished respiratory chain activity and widespread defects in mitochondrial gene expression

“…The copyright holder for this preprint (which this version posted June 1, 2020. ; https://doi.org/10.1101/2020.05.31.126607 doi: bioRxiv preprint overload-triggered opening of the mitochondrial permeability transition pore (PTP) and cell death (25). To explore the potential influence of Afg3l2 deficiency on mitochondrial calcium dynamics, we first tested whether Afg3l2 regulates the abundance of EMRE or other MCU subunits by co-expressing transgenes encoding tagged forms of these proteins, including EMRE, MICU1, and MCU along with the AFG3L2 RNAi transgenes using the da-Gal4 driver (26). We observed a significant increase in the abundance of Emre protein upon knockdown of Afg3l2, consistent with the conclusion that Afg3l2 is a negative regulator of Emre (Supplemental Figure 9A).…”

“…Calcium uptake assays were performed as described previously (26). Briefly, 200 µg of freshly isolated mitochondria from 9-day old pupae were incubated in respiration buffer (250 mM sucrose, 10 mM MOPS-Tris (pH 7.4), 5 mM glutamate, 2.5 mM malate, 5 mM Pi, 0.01 mM EGTA and 0.001 mM of Oregon green BAPTA 6F (Thermo Fisher)).…”