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First published online December 21, 2005
doi: 10.1242/10.1242/jcs.02719

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Single-molecule analysis of cadherin-mediated cell-cell adhesion

¹ Department of Chemical and Biomolecular Engineering, The Johns Hopkins University, 3400 N. Charles Street, Baltimore, MD 21218, USA
² Department of Chemical Engineering, University of Florida, Gainesville, FL 32611, USA

View larger version (70K): [in a new window]   Fig. 1. Principle of single-molecule force spectroscopy measurements in living cells. (A) Schematic of the molecular force probe (MFP) used here to probe cadherin-mediated cell-cell interactions at the single-molecule level. (B) Phase-contrast microscopy of cadherin-expressing CHO cells deposited on a cantilever. (C) Phase-contrast microscopy of adherent CHO cells attached to the end of a cantilever. Scale bars, 15 µm (B); 10 µm (C).

View larger version (25K): [in a new window]   Fig. 2. Force-distance spectra for cadherin/cadherin bonds. Typical force-distance curves during the forced de-adhesion of two apposing E-cadherin cells at a constant cantilever reproach velocity of 25 µm/second. One cell is attached to the cantilever of the MFP (shown in Fig. 1); the other cell is plated on a culture dish. Arrows indicate the rupture of cadherin bonds. Only curves showing a single clear bond rupture were analyzed.

View larger version (33K): [in a new window]   Fig. 3. Interaction between apposing cells are specific and involve cadherins. (A) Force-distance curves for cells with and without either EDTA or anti-E-cadherin function-blocking monoclonal antibodies (ECCD-1). Inset shows the force peak, from which rupture force (height of the peak) and loading rate (reproach velocity of the cantilever times the rate of increase before rupture) are obtained. (B) Distribution of N-cadherin cell aggregates, as assessed by flow cytometry. This graph shows that the biotinylation process does not change the distribution of cell aggregates and, therefore, the functionality of cadherins is unaffected by biotinylation. Solid bars represent conditions where EDTA is added to the solution; hatched bars represent conditions where calcium is added. Blue bars represent non-biotinylated cells; red bars represent biotinylated cells. (C) Phase-contrast micrograph of cells in different aggregation states. Bar, 10 µm.

View larger version (23K): [in a new window]   Fig. 4. Experimental and theoretical rupture force distribution of a cadherin-cadherin bond and heterotypic cadherin-cadherin interactions. (A) Experimental (white) and theoretical (black) histograms of rupture forces to break a single N-cadherin-N-cadherin bond connecting two apposing cells at a cantilever reproach velocity of 5 µm/second. Monte Carlo simulations, which were conducted using the Bell's model unstressed off-rate and reactive compliance extracted from the plot shown in Fig. 6A, show the consistency of the experimental data with the Bell's model predictions and further indicate that only one single type of bond is analyzed. (B) Representative force-distance curves for the detachment between two E-cadherin cells (top curve) and between an E-cadherin cell and an N-cadherin cell (bottom curve).

View larger version (100K): [in a new window]   Fig. 5. Rupture force of a single cadherin-cadherin bond as a function of reproach velocity. (A) Distribution of rupture forces to break a single E-cadherin-E-cadherin bond between apposing cells subjected to different reproach velocities, representing at least 500 cell-cell contacts at each velocity. (B) Distribution of rupture forces to break a single N-cadherin-N-cadherin bond at different reproach velocities, representing at least 500 cell-cell contacts at each velocity. Immunofluorescence micrographs of N-cadherin-expressing CHO cells (C) and E-cadherin-expressing CHO cells (D). Bar, 10 µm.

View larger version (19K): [in a new window]   Fig. 6. Single-bond analysis of cadherin-mediated cell-cell adhesion. (A) Mean rupture force of a single bond as a function of loading rate. Curve fitting with Bell's model allows for the computation of the unstressed dissociation rate, k0off and the reactive compliance, xß, of a bond. (B) Schematic of the intermolecular potential of interaction between E-cadherin pairs and N-cadherin pairs on apposing cells. This energy potential is qualitatively based on data shown in A. Blue represents the inner barrier potential at high loading rates; green represents the outer barrier potential at low loading rates for the dissociation of a single E-cadherin/E-cadherin bond between cells; red represents the single barrier potential to be overcome for the dissociation of a single N-cadherin-N-cadherin bond. The width of each potential well is taken as the reactive compliance.