Revealing Early Steps of ₂₁ Integrin-mediated Adhesion to Collagen Type I by Using Single-Cell Force Spectroscopy

Supplemental Materials

This article contains the following supporting material:

• MovieS1 - Phase contrast movie of a CHO-A2 cell approaching the collagen surface. Through z-piezo movement, the cell was lowered onto the collagen surface until a contact force of ~1nN was reached. Subsequently, the piezo position was kept constant. Simultaneously to the time lapse recording, the online signal of the cantilever deflection was recorded (real-time scan). The deflection signal was used to compile a contact force vs. time plot shown in the animation in the lower panel. Within ~2 sec after reaching the predefined contact force, the cantilever deflection partly relaxed due to the delayed viscoelastic response of the cell. Consequently, the effective contact force decreased slightly during the first 2 sec of matrix constant.
• MovieS2 - Phase contrast time-lapse movie of a CHO-A2 cell in matrix contact during SCFS. Frames were collected every second for a total of 300 seconds. Establishment of cell-matrix contact is indicated by the movement of the cantilever into focus. During contact, the cell became “activated”, since a maximal detachment force of ~8 nN was recorded at the end of the contact period.
• Figure S1 - Resistance of integrin α2β1 integrin to trypsin treatment. To assess the effect of trypsin treatment on the cell surface expression of α2β1 integrin, CHO-A2 cell surface proteins were biotinylated prior to different periods of trypsin treatment and subsequently purified using the EZ-Link Sulfo-NHS Biotinylation Kit (Pierce Biotechnology). Samples containing equal amounts of total protein (lysate) or biotinylated surface proteins were analyzed by SDS-PAGE and Western blotting for the integrin α2 (Mouse monoclonal anti-α2 integrin, Chemicon) and β1 subunits (rabbit antiserum R322, kindly provided by Jyrki Heino).
• Figure S2 - No influence of cell contact history on SCFS adhesion measurements. Two to five force cycles for contact times of 5 or 30 sec and two force cycles for a contact time of 300 sec were performed per CHO-A2 cell. Between force cycles the cell was allowed to recover for 2 to 3 minutes. Maximal detachment forces were averaged for all force curves recorded during the first, second, third, fourth or fifth cycle, respectively. Detachment forces showed no tendency towards increasing or decreasing values with higher numbers of force cycles.
• Figure S3 - Mechanical stability of the collagen matrix. (A) Detachment force curve for a strongly adherent cell (f_max> 15nN). (B) The cell contact area on the matrix imaged by AFM contact mode after force measurement. The matrix appeared undamaged by the cell adhesion and detachment process.
• Figure S4 - Monitoring the cell-matrix contact area during the force cycle. (A) Stills from a phase contrast movie displaying a single cell attached to the end of a tipless cantilever during the contact phase of a force cycle. Stills correspond to frames collected at the indicated time points during a total contact period of 300 sec (top row). The focal plane was chosen close to the collagen surface to contain the cell/substrate contact area. From the phase contrast images, cell contours were determined and the cell shape extracted using Photoshop (Adobe Systems Inc). The cell shapes (lower row) were thresholded and used to determine the cell/matrix contact area using ImageJ. The percentages indicate the contact area at the corresponding time point relative to the contact area after 5 sec of contact. (B) Quantification of cell/substrate contact area from three time-lapse movies recorded over 600 sec of contact time. Over the entire time course, the cell/matrix contact area fluctuated by up to 15% but did not increase overall.
• Table S1 - Number of cells (#) analyzed and number of force curves (n) generated.