AFM measurements and lipid rearrangements: evidence from red blood cell shape changes†
Application of force to echinocytes during atomic force microscopy measurements was shown to be able to convert the cells to stable discocyte shapes. The echinocyte shape is associated with a relative excess of the area of the outer leaflet of the cell membrane; the AFM measurements are therefore associated with a change in the relative areas of the inner and outer membrane leaflets. It was hypothesized that localised damage in the lipid bilayer that is caused by an AFM tip can permit the lipids to flip-flop between the two membrane leaflets, thus changing their relative areas. The conditions in which AFM measurements on cells could induce shape changes were investigated both experimentally and by modelling. The relative area change of the membrane leaflets, attributed here to lipid movement, was characterised in terms of the membrane energy levels; membrane energy was calculated using a version of the area-difference-elasticity model that was applied to predetermined shapes, rather than being used to generate shapes as solutions found at the energy minima. Shapes were generated by rotation of Cassini ovals with a superimposed undulation in order to generate spikes similar to those of the echinocytes. The membrane energy was considered as a function of the membrane curvature, the area difference between the two membrane leaflets, and the deformation of the cytoskeleton. This led to the conclusions that the minimisation of the membrane energy causes the lipid translocation, with the relaxation of the cytoskeleton being a significant driving force.