Fig3. MICU1 contributes to cooperative activation of mtCU.

(A) Double logarithmic plots of initial Ca²⁺ uptake rates vs. [Ca²⁺]_c in Ctrl and MICU1-KD hepatocytes (left) and stable HeLa cells in the presence (middle) and absence of Mg²⁺ (right). Slope of linear fit for each data set is shown (n=3). (B) [Ca²⁺]_c dose-responses of the fractional mitochondrial Ca²⁺ uptake in Ctrl, RESCUE and ΔEF-RESCUE HeLa cells. Data points from individual recordings (≥3 for each Ca²⁺ dose) and a sigmoid (logistic) fit to each data set are shown. The sigmoid fit for MICU1-KD cells from Fig2D is shown as a reference (red). (C) [Ca²⁺]_c (top) and ΔΨ_m (bottom) time courses showing the mitochondrial clearance of a 100 μM CaCl₂ pulse (100Ca) and subsequent FCCP-induced Ca²⁺ release in suspensions of permeabilized HeLa cells. Inset: mean initial Ca²⁺ release ([Ca²⁺]_c rise) rates from recordings where by the time of FCCP addition [Ca²⁺]_c dropped below 1.2 μM (Means±S.E., n=3). Rates obtained from recordings in the presence of RuRed (3 μM) added 30 s before FCCP are shown with open symbol. (D) Left: Instantaneous rate of FCCP-induced Ca²⁺ efflux at each [Ca²⁺]_c (0.5μM binning) obtained via differentiation of the initial period of the [Ca²⁺]_c rises (n=7). Right: Instantaneous Ca²⁺ release rates extrapolated from compilation of differentiated sigmoidal (logistic) fits. Predicted 0 crossings (the effective thresholds) are 937 and 185 nM for Ctrl and MICU1-KD, respectively.