Fig 1: Expression of MCUWT and MCU mutants in HeLa cells A, BHis-tagged MCUWT and MCU ? NTD were expressed in HeLa cells, and the expression of the proteins was detected with anti-His (A) and anti-MCU (B) antibodies, respectively.CLocalization of MCUWT and MCU ? NTD was examined by confocal imaging after co-transfection of GFP-tagged MCUWT or MCU ? NTD with mito-DsRed. The merged images indicated that MCU ? NTD is localized in the mitochondria, similar to MCUWT. Scale bars, 10 ?m.DExpression profiles of MCU regulatory proteins. Twenty micrograms of the lysates with or without dithiothreitol (DTT) were subjected to SDS–PAGE and immunoblotted using the indicated antibodies. The results revealed that the expression levels of the indicated proteins did not change in MCU ? NTD-overexpressing HeLa cells.EInteraction of MCU mutants with MICU1 and 2. MCU ? NTD, MCUS 92A and MCUK 180A with MICU1 and MICU2 bind to MICU1 and MICU2 as MCUWT does. After expression of Flag-tagged MCUWT, MCU ? NTD, MCUS 92A and MCUK 180A in MCU-KD HeLa cells, co-immunoprecipitation assay was performed. The precipitates were subjected to SDS–PAGE and immunoblotted with the indicated antibodies.
Fig 2: Electrostatic interactions between MCU and MICU1.(A) Modulation of MCU-MICU1 complex stability by ionic strength. WT MCU and MICU1 were expressed in MCU/EMRE-KO cells, and CoIP experiments were performed in the presence of 50, 150, or 500 mM of NaCl. The IP signal of MCU was normalized to that of MICU1, with the ratio presented in the bar chart. (B) The effect of MICU1 Arg mutations on MCU binding. (C) A CoIP experiment testing if MCU and MICU2 form complexes. MICU2 was FLAG-tagged to precipitate WT MCU in MCU/EMRE-KO cells. *p<0.05.
Fig 3: The impact of D261 or E264 mutations on MICU1 binding.FLAG-tagged WT MICU1 was used to pull down various MCU mutants co-expressed in MCU/EMRE-KO cells.
Fig 4: Loss of control over mitochondrial Ca2+ uptake turns regeneration signals to death signals in MICU1-deficient hepatocytes.Partial hepatectomy, a surgical model for extensive tissue injury/loss in the liver, reprogrammes the fully differentiated hepatocytes in the remnant liver to proliferate in order to regenerate liver parenchyma. Reprogramming involves the synchronized priming of hepatocytes by growth factors and pro-inflammatory cytokines to obtain replicative competence. These mediators engage a broad range of intracellular signals, including Ca2+, which in addition to driving cytosolic effectors, can propagate to the mitochondrial matrix and control metabolic enzyme activities to support the proliferative response. Mitochondrial Ca2+ uptake is mediated by the Ca2+-gated mitochondrial Ca2+ uniporter complex. Ca2+ gating occurs through MICU1 (yellow trapezoid), which associates with the MCU pore (grey cylinder) from the intermembrane space side to set the threshold for Ca2+ activation. As a result, mitochondrial Ca2+ uptake occurs largely through local, high [Ca2+] elevations, whereas smaller events and global Ca2+ fluctuations are repelled (left-side purple arrows). Upon loss of MICU1 control over the Ca2+ uptake threshold, smaller [Ca2+] elevations can activate uniporter-mediated uptake (right-side purple arrows). Ca2+ signalling events occurring after PHx can thereby lead to mitochondrial Ca2+ overload to enhance permeability transition pore (PTP) opening and switch the cell's fate from proliferation to a death track. Large-scale hepatocyte death will overwhelm the functional capacity of the remaining liver cells resulting in organ failure (dashed arrow). Pharmacological inhibition of the PTP by NIM811 rescues mitochondrial competence to support proliferation even under the higher Ca2+ load in MICU1-ablated cells (thick blue arrow), which may provide the stimulus for accelerated cell proliferation under these conditions.
Fig 5: In human aortic endothelial cells (HAECs), levels of MCU (mitochondrial Ca2+ uniporter) subunit proteins are not altered by sex hormones. A, Representative immunoblots for MCU subunits MCUa, MCUb, EMRE, and MICU1, as well as the housekeeping protein COXIV (control), in mitochondrial fractions of HAECs treated with testosterone, estradiol, or diluent (nontrearted [NT]) for 72 hours. B through E, Quantification of levels of the (B) MCUa, (C) MCUb, (D) MICU1, and (E) EMRE proteins, normalized to COXIV. N=8 (B), 12 (C), 11 (D), 4 (E) biological replicates. Analysis by Friedman test. COXIV indicates cytochrome c oxidase subunit IV; EMRE, essential MCU regulator; Estr, estradiol; MCUa, mitochondrial calcium uniporter a; MCUb, mitochondrial calcium uniporter b; MICU1, mitochondrial calcium uptake 1; NT, nontrearted; Test, testosterone.
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