JCS024877 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Cadherin expression profiles of differentiated and proto-MFs. Using flow cytometry, expression levels of (A) α-SMA, (B) N-cadherin and (C) OB-cadherin were compared between differentiated MFs (black) and proto-MFs (dark grey). Controls using secondary antibodies only are displayed in light grey. Events=counts×100.

• Supplemental Figure S2 -

  Fig. S2. AFM detects rupture of cadherin bonds. (A) AFM cantilevers supplied with recombinant cadherin dimers or with MFs were contacted with cadherin dimers carpets and MF monolayer and then re-lifted (here demonstrated for cell-cell interaction). In series of preliminary experiments we determined these optimum parameters: loading force of 3 nN, cantilever approach/retraction velocities of 0.1-1.0 µm/second and contact times of 1-60 seconds. (B) When the cantilever is lifted after contact formation, adhesion events lead to deflection that is plotted over the distance z between cantilever base and substrate. In experiments using cadherin-grafted AFM cantilevers, we typically obtained one to two rupture events that appear as ‘jumps’, whereas detaching two MFs created a saw-tooth profile with multiple jumps. In all setups, jumps exhibit different heights and always occur in one single step. In the presence of EGTA, no interaction occurs between cantilever and substrate, demonstrating Ca²⁺ dependence of bond formation. (C) The height of each jump in the force-distance profile represents bond strength (pN); the area included by the retraction curves and the cantilever zero-deflection level represents the work of total cell detachment (pN^.µm). The slope of the curve preceding a single rupture event represents the mechanical loading of the single bond.

• Supplemental Figure S3 -

  Fig. S3. Effect of anti-cadherin peptides on intercellular MF adhesion. Differentiated MFs (A,C,E) and proto-MFs (B,D,F), grown on tipless AFM cantilevers were contacted with the same respective cell type grown in monolayer for 2 seconds with constant approach velocity (0.1 µm/second) and loading force (3 nN). (A,B) Typical force distance curves are displayed for each configuration; arrows indicate positions where bond rupture occurs in a ‘jump’. Red profiles indicate typical interaction two MFs in the control condition, pink lines represent contact formation in the presence of blocking peptides directed against the cadherin type expressed on the respective cell type, and red lines indicate contact formation in the presence of blocking peptides directed against the cadherin type that is not expressed on the respective cell surface. (C-F) The number of rupture jumps preceding total cell detachment was extracted from force-distance curves and displayed in histograms for (C) differentiated MFs in the presence of anti-OB-cadherin, (D) proto-MFs in the presence of anti-N-cadherin, (E) differentiated MFs in the presence of anti-N-cadherin, and (F) proto-MFs in the presence of anti-OB-cadherin.

• Supplemental Figure S4 -

  Fig. S4. The bond strength of native cadherins in living MFs increases with increasing contact time. Differentiated MFs grown on tipless AFM cantilevers were contacted with differentiated MFs grown in monolayer for different contact times (2-60 seconds) with constant approach velocity (0.1 µm/second) and loading force (3 nN). (A) All obtained rupture forces are displayed as Gaussians fits of histograms (n≥5000 per contact time). Overlaying all Gaussian fits obtained for each contact time demonstrates increasing formation of a third force peak with increasing contact time at decreasing amplitude of the first peak. Gaussian fittings obtained after 3 seconds (C), 5 seconds (D), 10 seconds (E) and 60 seconds (F) of contact time are normalized to the total number of events obtained after 2 second contact time (B) to facilitate comparison. The dashed lines represent Gaussian fittings of single peaks, which are included in each total data set.