Fig. 2
From: MICU1 controls cristae junction and spatially anchors mitochondrial Ca2+ uniporter complex
ψmito and the poly-lysine domain of MICU1 are required for IBM localization. a Schematic representation of MICU1 mutants used in this work. (MTS→mitochondrial targeting sequence; KKKK→poly-lysine domain; EF-Hand→Ca2+ binding domain; C-Helix→C-terminal hexamerization domain). b Representative images of SIM time-lapse experiment using HeLa cells transiently transfected with MICU1-YFP (green) and EMRE-mCherry (magenta) after T = 0 and T = 270 s. Cells were pre-incubated with oligomycin A (2 µM) and antimycin A (4 µM) was added at T = 90 s. c Line plots of MICU1 (green line) and EMRE (magenta line) of regions in between the white arrows in b confirm the redistribution of MICU1 into the IMM after addition of antimycin A. Differences between oligomycin A and oligomycin A plus antimycin A traces were measured from T = 50 s. d Time course of the MICU1 IBM association index over time after pre-incubation with oligomycin A with (green) or without (black) exposure to antimycin A starting at T = 90 s (nControl = 7; nOligomycin/Antimycin = 9. Data are shown as the mean ± SEM. e Corresponding tetramethylrhodamine methyl ester perchlorate (TMRM) fluorescence, which indicates ψmito. Cells were pre-incubated with oligomycin A and exposed to antimycin A (green) or DMSO (black) at T = 90 s (nAntimycin = 8; nAntimycin/Oligomycin = 9). A significant drop in ψmito after addition of antimycin A correlates with the drop in MICU1 IBM association index. Data are shown as the mean ± SEM. f Representative dual-SIM images of HeLa cells stained with MTR (magenta) and expressing MICU11–140-cpEGFP (green, upper panel) or MICU11–70-cpEGFP (green, lower panel). g Respective statistical analyses of the IBM association index of MICU11–140 and MICU11–70. The sub-mitochondrial distribution of MICU11–140 and MICU11–70 in the IBM and the CM was determined with the IBM association index. Horizontal lines represent the median, the lower and upper hinge show respectively first quartile and third quartile, and lower and upper whisker encompass minimal and maximal values. Images and analyses were obtained from n = 9 independent experiments, which assessed a total of 45 cells. *P < 0.05 vs. respective control conditions carried out with unpaired double-sided T-test. Source data are provided as a Source Data file
