Abstract
Differential binding between cadherin subtypes is widely believed to mediate cell sorting during embryogenesis. However, a fundamental unanswered question is whether cell sorting is dictated by the biophysical properties of cadherin bonds, or by broader, cadherin-dependent differences in intercellular adhesion or membrane tension. This report describes atomic force microscope measurements of the strengths and dissociation rates of homophilic and heterophilic cadherin (CAD) bonds. Measurements conducted with chicken N-CAD, canine E-CAD, and Xenopus C-CAD demonstrated that all three cadherins cross-react and form multiple, intermolecular bonds. The mechanical and kinetic properties of the heterophilic bonds are similar to the homophilic interactions. The thus quantified bond parameters, together with previously reported adhesion energies were further compared with in vitro cell aggregation and sorting assays, which are thought to mimic in vivo cell sorting. Trends in quantified biophysical properties of the different cadherin bonds do not correlate with sorting outcomes. These results suggest that cell sorting in vivo and in vitro is not governed solely by biophysical differences between cadherin subtypes.
Highlights
Cadherins are cell-surface adhesion molecules that mediate calcium-dependent intercellular adhesion in all solid tissues
Homophilic Cadherin Bonds Exhibit Similar Strengths and Dissociation Rates—We measured homophilic binding between the Fc-tagged extracellular domains 1–5 of three classical cadherins: namely, chicken neural cadherin (N-CAD), Xenopus C-CAD, and canine epithelial cadherin (E-CAD)
The bin size h is estimated by minimizing the integral of the mean square errors (MSE) [43] and is approximated to be h ϭ 3.5 ϫ ϫ nϪ1/2, where is the standard deviation of the distribution, and n is the total number of data points (n ϳ 400, in these studies)
Summary
CHO-K1 cells stably expressing wild type cadherin were cultured in Glasgow modified Eagle’s medium with 10% fetal bovine serum. The E-CHO cells were stained with anti-E-CAD antibody (BD Biosciences) and with PE-Cy5-conjugated antimouse IgG antibody (Santa Cruz Biotechnology, Santa Cruz, CA) in PBS with 0.5 mM EDTA and 1% BSA at 4 °C. The sample was mounted onto the AFM stage and incubated with protein solution (0.3 M cadherin in PBS) for 1 h at room temperature. The second type of measurement determined the bond lifetimes using the constant force mode. Fits of Equation 4 (or a superposition of exponentials) to plots of the survival probability versus time at a constant force fi give the number of bound states with rupture forces greater than fi and the dissociation rates. The dissociation rates are related to the lifetimes ti by ti ϭ 1/ki
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