Imaging dishes

The proximity ligation assay (Duolink™, Sigma-Aldrich) was performed according to the manufacturer’s instructions. Briefly, HeLa cells were grown on imaging dishes (Ibidi), then fixed with ice-cold methanol for 7 min on ice. Cells were incubated with 3% BSA in DPBS for 1 h and then with primary antibodies overnight at 4 °C. Subsequently, cells were incubated with oligonucleotide-labeled secondary antibodies, followed by ligation, DNA amplification, and probe hybridization. Resulting signals were visualized by confocal microscopy.

HAP1^IFN‐γR2KO cells were seeded onto imaging dishes (Ibidi 81156) in IMDM media and transfected with GFP‐tagged IFN‐γR2WT or IFN‐γR2^TM. RICS acquisitions were recorded on a commercial LEICA SP8 3X STED SMD confocal microscope (Leica Microsystems, Manheim, Germany), with an HCX PL APO 63x/1.2NA CORR CS2 water immersion objective and using a WLL as pulsed laser source. Relative power, as it appears in the LAXs software, was always below 2%. The images shown were representative of the experiments used for quantification out of a larger set of time lapses that were also analyzed for statistical purposes. Diffusion analysis by RICS was examined using SimFCS 4 software (G‐SOFT Inc.), as previously described in ref. [32 ]. Point spread function was determined as described elsewhere.^[32] RICS images series (256 × 256 pixels) were taken using either an 8 or 4 µs dwell time with no difference in the diffusion yielded between them. Each time‐lapse was taken for 200–300 total frames. From each full‐frame time‐lapse, a smaller region of interest was selected (32 × 32 pixels), and the diffusion coefficient was obtained by fitting the experimental 2D autocorrelation function to a single diffusion mode. The 2D autocorrelation map was then fitted to obtain a surface map, employing the characterized waist value and the appropriate acquisition values for line time and pixel time.

C2C12 myoblasts stably expressing mCherry‐GFP‐mtFIS1_101‐152 were seeded on imaging dishes (Ibidi, Gräfelfing, Germany) and treated with either 10 μM of CCCP or 20 μM of AMPK activator 991. Following treatment, cells were washed twice with PBS and fixed in 3.7% of formaldehyde with 200 mM of HEPES (pH 7.0) for 10 minutes. After fixing, cells were washed and incubated for 10 minutes in DMEM supplemented with 10 mM of HEPES (pH 7.0), and then washed with PBS before mounting with Prolong gold mounting solution containing 4′,6‐diamidino‐2‐phenylindole (DAPI; ThermoFisher Scientific, Leicestershire UK). Images were taken using a Crest X‐Light spinning disk system coupled to a Nikon Ti‐E base, 60x/1.4 NA (CFI Plan Apo Lambda) air objective and Photometrics Delta Evolve EM‐CCD. For GFP, excitation was delivered at λ = 458‐482 nm using a Lumencor Spectra X light engine, with emitted signals detected at λ = 500‐550 nm. For mCherry, the wavelengths used for excitation and detection were λ = 563‐587 nm and λ = 602‐662 nm, respectively.