Nanomechanical Investigation of Soft Biological Cell Adhesion using Atomic Force Microscopy

Abstract

Mechanical coupling between living cells is a complex process that is important for a variety of biological processes. In this study the effects of specific biochemical treatment on cell-to-cell adhesion and single cell mechanics were systematically investigated using Atomic Force Microscopy (AFM) Single Cell Force Spectroscopy. Functionalised AFM tipless cantilevers were used for attaching single suspended cells that were brought in contact with substrate cells. Cell-to-cell adhesion parameters, such as maximum unbinding force (F max ) and work or energy of detachment (W D ), were extracted from the retraction force-displacement (F-d) curves. AFM indentation experiments were performed by indenting single cells with a spherical microbead attached to the cantilever. Hertzian contact model was applied to determine the elastic modulus (E). Following treatment of the cells with neutralising antibody for epithelial (E)-cadherin, F max was increased by 25%, whereas W D decreased by 11% in response to a 43% increase in E. The results suggest that although the adhesion force between cells increased after treatment due to higher ligand-receptor binding, the energy of adhesion was decreased due to the reduced displacement separation as manifested by the loss of elastic deformation. Conclusively, changes in single cell mechanics are important underlying factors contributing to cell-to-cell adhesion and hence cytomechanical characterization is critical for cell adhesion measurements.

Quantifying cellular mechanics and adhesion in renal tubular injury using single cell force spectroscopy

“…Altering the expression of the adhesion protein does not absolutely indicate that the function of the cell-to-cell adhesion will correspondingly change. 16 The involvement of binding partners linked to the cellular architecture adds to the complexity of surface molecular interactions and hence cell adhesion is affected by changes in binding affinity, bond number and mechanical deformation. 21 In the current study, the TGF-β1 evoked reduction in E-cadherin expression was matched by a decrease in functional tethering between cells, as shown by the reduction in the number of TREs after TGF-β1 13 treatment.…”

“…The criteria and assumption for applying this model to biological material are described elsewhere. [14][15][16][17] However, it has been well recognized that the Hertz model often overestimates the elasticity of cells due to substrate effects. Alternatively, the classic Hertz model which was modified by Chen to take account of the influence of the substrate to the measured elasticity may be applied for modelling the cell indentation tests.…”

Determination of work of adhesion of biological cell under AFM bead indentation

“…For the adhesive contact between a cell and a polystyrene bead, the radius of MIN6 cell is typical of 12.5 μm, and the elastic modulus of cell is in the order of several hundred Pascal. The previous studies have found the value of is about 500Pa by using Hertz contact theory to fit the F-D curve of an AFM indented MIN6 cell (Siamantouras et al, 2014b). The work of adhesion ∆γ ranges from 20 to 100 μJ/m 2 , and here we assume ∆γ to be 50 μJ/m 2 (Sirghi et al, 2008).…”

“…Recording of F-d curves was conducted in liquid media (CO 2 free DMEM). To perform single cell indentation experiments, a spherical microbead (11 μm in diameter) was manually attached on the cantilever and the spring constant was then determined to be 0.059 N/m by using the thermal noise method after attachment (Siamantouras et al, 2014b). The depth of indentation was calculated by subtracting the deflection of the cantilever from the displacement of the piezo-actuator for a given spring constant (JPK Data Processing, Berlin, Germany).…”

“…The data used for testing the theoretical model were extracted from the recently reported AFM indentation experiments of pancreatic cells (Siamantouras et al, 2014a), in which Hertz contact model was applied to analyse F-d curves for calculating the Young's modulus (E) between two sets of data with different elasticity. In this paper, we have used MIN6 cells cultured for 48h with low glucose which are named as Sample A and cells cultured in 48h +R568 are named as Sample B (Siamantouras et al, 2014b).…”