μ dish35 mm grid 500

Force indentation experiments were carried out continuously on a Nanowizard® II or III AFM (JPK Instruments, Berlin, Germany) while scanning laterally across the sample referred to as force mapping. Before use cantilevers (MLCT; Bruker AFM Probes, Camarillo, USA) were plasma cleaned (30 s, Argon) and incubated with PBS⁻ containing 2.5 mg/mL concanavalin A-FITC conjugate (Sigma-Aldrich, Steinheim, Germany) for 1.5 h to establish a strong contact between the indenter and the cell membrane for membrane-tether pulling upon retraction from the cell surface. Afterwards, the cantilever was also calibrated using thermal noise method53 . Prior to indentation experiments cells were seeded on Petri dishes (μ-Dish^35 mm Grid-500; ibidi, Martinsried, Germany) and manipulated as desired. After cells reached confluency Petri dishes were mounted on an inverted microscope and kept at 37 °C. Cells were indented up to a force of 1 nN. After a dwell time of 0.5 s the tip was retracted from the cell surface pulling out membrane nanotubes (tethers) from the plasma membrane. The pulling velocity was set to 2 μm/s. Indentation curves were analysed by applying an extended tension model27 39 (link) (vide supra) while tether forces were determined directly from retraction curves (Fig. 1b).

Madin-Darby canine kidney cells (strain II, MDCK II; Health Protection Agency, Salisbury, UK) were maintained in minimum essential medium (MEM) with Earle’s salts and 2.2 g/L NaHCO₃ supplemented with 4 mm l-glutamine and 10% fetal calf serum at 37 °C in a 5% CO₂ humidified incubator. Cells were grown to confluency, released from culture flasks using trypsin/EDTA (0.05%/0.02%) and subcultured weekly. Medium additionally contained penicillin (0.2 mg/mL), streptomycin (0.2 mg/mL) and HEPES (15 μm) during experimentation.
For ezrin inhibition experiments with ezrin inhibitor NSC 668394, cells were seeded on Petri dishes (μ-Dish^35 mm Grid-500; ibidi, Martinsried, Germany) and grown to confluency. NSC 668394 (Merck Millipore, Molsheim, France) was dissolved in DMSO. An appropiate amount of this stock solution was added to cell culture media (final concentration: c_{NSC 668394} = 250 μm) and cells were incubated for 3 h. For control measurements cells were incubated for 3 h with media containing the same amount of DMSO without NSC 668394.

Su9-RFP MEFs, Drp1-knockout Su9-RFP MEFs^{27 (link)}, and HeLa cells (ATCC CCL-2) were grown on glass-bottomed 35-mm culture dishes (Matsunami, Tokyo, Japan) for conventional observation and a μ-Dish 35 mm Grid-500 (Ibidi, Martinsried, Germany) for CLEM in Dulbecco’s modified Eagle’s medium (DMEM) with 10% foetal bovine serum (FBS) in an incubator with 5.0% CO₂ at 37 °C. HeLa cells were transfected with CellLight Mitochondria-GFP or Mitochondria-RFP BacMam2.0 (Thermo Fisher Scientific, Waltham, MA, USA) to visualise mitochondria at 60–70% confluence in complete medium according to the manufacturer’s instructions.