Characterization of cholesterol-depleted or -restored cell membranes by depth-sensing nano-indentation

Cholesterol is a major component of eukaryotic cell membranes. It is well-accepted that cholesterol depletion triggers a complicated cascade of biochemical reactions which may affect many cell processes. However, the effect of cholesterol depletion on the deformability of cell membranes is still controversial. In this study, depth-sensing nano-indentation is performed on the lamellipodium of adherent NIH-3T3 fibroblast cells with normal, depleted, and restored membrane cholesterol contents. By extracting data from contact stiffness measurement, nano-mechanical characterizations are focused at a depth within the superficial 20 nm of the tested cells. Our results show that cholesterol depletion indeed decreases membrane stiffness, while the membrane stiffness decreases exponentially with the increase of cholesterol-depletion time. In addition, the effect of cholesterol restoration following depletion is further examined, showing that cholesterol restoration reverses the effect of cholesterol depletion on both cellular morphology and membrane stiffness. This is the first study, focused on nano-mechanical characterization of cellular outermost layers, demonstrating the effect of altered cholesterol content on the stiffness of cell membranes.