Quantitative investigation of calcium-sensing receptor activation on beta-cell adhesion and mechanics.

Aim: Activation of the extracellular calcium-sensing receptor (CaSR) on beta cells increases expression of the adhesion protein epithelial (E)-cadherin. Glucose-responsiveness relies on cell-cell interactions. In this study we quantified changes in cell-cell tethering and elasticity in response to the calcimimetic R568 in clonal beta cells. Methods: Cell-cell adhesion and single cell indentation experiments were performed by atomic force microscopy based force spectroscopy (AFM-FS) on MIN6 cells +/- R568 (1μm) at 37oC. The AFM-FS was fitted with a bead-attached cantilever to indent single cells and calculate cell elasticity, i.e. Young’s modulus, from force-displacement (F-d) curves. Results: The calcimimetic R568 (1μM) increased the maximum unbinding force between cells by 29% and detachment energy by 37% (12cells, n=3, p<0.001). To assess the effects of viscoelastic deformation on cell adhesion F-d curves were performed after increasing the speed of retraction (5-to-12.5μm/sec). As the pulling speed increased, the energy of detachment required to completely separate two adherent cells increased by 40% and maximum unbinding force increased by 14% (10cells, n=3). Applying the Hertz contact model for elastic indentation to F-d curves recorded during indentation of single cells, information on elastic or Young’s modulus was extracted. The results indicate that R568 (1μΜ) decreased the elastic modulus by 34% (30cells, n=3, p<0.001). Conclusion: Adhesion and indentation measurements suggest that viscoelastic deformation has a significant influence on the adhesion energy of detachment between adherent beta cells. Increased CaSR evoked E-cadherin ligation only partially explains the increased the work of separation energy of detachment, which also depends on the viscoelastic/mechanical properties of the cell. This work is supported by grants from Diabetes UK (BDA: 12/0004546 and 09/0003913).