Fixed endothelial cells exhibit physiologically relevant nanomechanics of the cortical actin web

It has been unknown whether cells retain their mechanical properties after fixation. Therefore, this study was designed to compare the stiffness properties of the cell cortex (the 50-100 nm thick zone below the plasma membrane) before and after fixation. Atomic force microscopy was used to acquire force indentation curves from which the nanomechanical cell properties were derived. Cells were pretreated with different concentrations of actin destabilizing agent cytochalasin D, which results in a gradual softening of the cell cortex. Then cells were studied 'alive' or 'fixed'. We show that the cortical stiffness of fixed endothelial cells still reports functional properties of the actin web qualitatively comparable to those of living cells. Myosin motor protein activity, tested by blebbistatin inhibition, can only be detected, in terms of cortical mechanics, in living but not in fixed cells. We conclude that fixation interferes with motor proteins while maintaining a functional cortical actin web. Thus, fixation of cells opens up the prospect of differentially studying the actions of cellular myosin and actin.