Effect of cholesterol depletion on the viscoelastic properties of alveolar epithelial cells assessed by Atomic Force Microscopy in large deformation

The alveolar epithelium plays the role of the first physical barrier to prevent the internalization of harmful molecules or germs contained in inhaled air. This function is notably assured by the maintenance of the mechanical integrity at the cellular scale of the alveolar epithelial cell plasma membrane. A major component and organizer of the plasma membrane, well known to play a key role in mechanical properties of the cell and its plasma membrane, is the cholesterol. But, nowadays there are few data on the mechanical properties of alveolar epithelial cells in normal and in cholesterol-depletion conditions. Thus, in this work, the viscoelastic properties of adherent living alveolar epithelial cells are assessed by a relaxation mechanical assay performed with Atomic Force Microscopy. Concretely, during one minute of indentation in cell, the cantilever piezo height is fixed while the relaxing force is recorded. The first part of the force-distance AFM curve is fitted by a Hertz-based model and the second part of the force-time AFM curve corresponding to relaxation phase is analyzed by a generalized Maxwell-Wiechert model at second order. AFM measurements were performed on A549 cells cultured for 24 h following 3 different culture conditions: (1) baseline condition, (2) Methyl-b-Cyclodextrin condition (MbCD); this drug, known to deplete cholesterol in the cell membrane and to disrupt lipid raft, allows to test the role of cortical membrane stiffness in mechanical properties of adherent epithelial cells, and (3) cytochalasin-D condition; this drug depolymerizes the actin cytoskeleton of the cells. The results show that (i) as expected, actin-filament depolymerization with cytochalasin-D decreases cell stiffness up to 50%, and (ii) after 1 hour of MbCD incubation, the A549 cell stiffness increases up to 80% as observed in other cell types. This study highlights the key role of cholesterol in the mechanical integrity of alveolar epithelial cells and offers a novel analysis model to extract cellular viscoelastic properties from AFM data. © 2017 Lavoisier.