Biocell

Stiffness of cell nucleus and cytoplasm was measured with a NanoWizard 4 AFM (JPK) instrument mounted on top of a Nikon Ti ECLIPSE microscope^{62 (link)}. The spring constant of the cantilevers was calibrated by thermal tuning using the simple harmonic oscillator model. The Hertz model was fitted to the approach curves to obtain the stiffness value using the JPKSPM Data Processing software (version 6.1.79). The cells were seeded on laminin-111-coated coverslips and a force curve on top of the nucleus and cytoplasm was acquired for each of the cells. The cells were kept at 37 °C using a BioCell (JPK) and maintained in a CO₂-independent medium (Thermo Fisher, category no. 18045088).

Adherent cells were cultured on 70% ethanol-cleaned glass slides in 6-wells culture plates, rinsed to remove unbound cells and fragments and mounted in a temperature-controlled chamber (Biocell, JPK Instruments, Berlin, Germany) set to 37 °C. Cells were indented with a JPK Nanowizard 1 AFM (JPK Instruments), using the force mode with a closed loop 15 μm range piezo. The AFM sits on an Axiovert 200 microscope equipped with a Colibri 2 diode illumination system (Zeiss, Oberkochen, Germany) and a CoolSnap HQ2 camera (Photometrics, Tucson, AZ, USA). A glass sphere of diameter 10 μm was glued by micromanipulation (using a homemade micropipette/biomembrane force probe setup) to a gold-coated triangle-shaped MLCT cantilever (Bruker Instruments, Billerica, MA, USA), using UV polymerizable glue (Dymax OP-29) in order to measure cell mechanics on similar scales as in the microindentation experiments. The decorated AFM cantilever was calibrated in situ prior to the experiments using the thermal noise method implemented in the JPK SPM control software and found to be 11.5 nN/μm, compatible with the nominal data provided by the manufacturer (10 nN/μm). The approach and retract speeds of the indenter were 1 μm/s over a distance of 5 μm and the maximal applied force was set between 3 and 6 nN. The acquisition frequency was set at 1024 Hz.

GPMV, PM patches and supported planar bilayers (SPB) formed by cell lipid extracts were scanned by AFM. Cell lipid extract samples were prepared on V-2 high quality scratch-free mica substrates (Asheville-Schoonmaker Mica Co., Newport News, VA, USA). A total of 180 μL assay buffer containing 3 mM CaCl₂ was added onto a 1.2 cm² freshly cleaved mica substrate mounted onto a BioCell (JPK Instruments, Berlin, Germany). Then, 80 μL sonicated 0.4 mM SUV formed from CHO lipid extract were added on top of the mica slip. BioCell temperature was gradually increased (5 °C every 5 min) up to 80 °C. Vesicles were left to adsorb and extend for 30 min keeping the sample temperature at 80 °C. A further 30 min were allowed for the samples to equilibrate at room temperature before performing five washing steps with calcium-free buffer in order to discard non-adsorbed vesicles and remove the remaining Ca²⁺ cations [34 (link)].