Erythrocyte attachment to substrates: determination of membrane tension and adhesion energy

Abstract

The adhesion of osmotically swollen erythrocytes to glass substrates is studied. An experimental set up is used, which allows one to carry out measurements with the same adherent cell at various compositions of the medium. The reflection interference microscopy is combined with the generalized Laplace equation describing the shape of the cell membrane. The latter equation, which accounts for the membrane bending elasticity, is solved numerically along with appropriate boundary conditions; the necessary values of some geometrical parameters are extracted from the interference data. The mathematical problem has a unique solution, which gives the values of the membrane tension, the pressure drop across the membrane, and the energy of adhesion. To reveal the physical origin of the observed cell-to-glass adhesion we carried out experiments with untreated (native) erythrocytes, as well as with trypsin-treated and glycophorin-treated erythrocytes. The former and the latter treatment, respectively, decreases and increases the outer surface electric charge of the cell. The comparison of the determined adhesion energies for untreated and trypsin-treated erythrocytes indicates that the adhesion could be attributed to the short-range electrostatic attraction due to the discreteness of the surface charge. The results for the glycophorin-treated erythrocytes show that at 20°C there is no cell-to-glass adhesion at all, whereas at 37°C adhesion is observed, but it is weaker than for untreated cells; this could be explained with the enhancement of the electrostatic double layer repulsion by the incorporation of additional electrically charged glycophorin molecules in the cell membrane. The developed mathematical and numerical procedure for solving the generalized Laplace equation can find application for interpreting the configurations of biological and model membranes (vesicles), both free and attached, and can bring information about physical parameters, such as membrane tension, difference in pressure or electric potential across the membrane, its bending elasticity and spontaneous curvature, energy of adhesion, etc.