Mlct cantilever

Experiments were performed using a ForceRobot300 (JPK) atomic force microscope. The MLCT cantilever (Bruker Corp.) was calibrated in the solution using the equipartition theorem, and a spring constant of ~50 pN·nm⁻¹ was usually obtained. All experiments were performed at room temperature in a Tris buffer of pH 7.4. The protein solution was first absorbed onto a glass coverslip and then was subjected to experiments after 10 minutes of incubation. The tip contacts the surface for hundreds of milliseconds and then retracts at a constant velocity. The pulling velocities for experiments were 400 nm·s⁻¹ and 1000 nm·s⁻¹, respectively. To ensure the data is from the real multistep unfolding events of βMT, not due to the background noise, only force-extension curves with stepwise rupture peaks whose cumulative ΔLc is ~8 nm (between 7 nm to 10 nm) were selected for further analysis. At least the ΔLc of one force peak is larger than 3 nm. Statistically, ~50 out of 1000 (~5%, 400 nm·s⁻¹ condition) βMT unfolding curve showed such multistep unfolding events.

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.

To measure the thickness of ECMs following wound scratch, AFM force spectroscopy and contact imaging were performed using a JPK NanoWizard II Bio AFM in combination with a Bruker MLCT cantilever. A Nanoworld Arrow TL-1 cantilever with bead attached was used for force spectroscopy.