Hypotonic Challenge of Endothelial Cells Increases Membrane Stiffness with No Effect on Tether Force.

Regulation of cell volume is a fundamental property of all mammalian cells. Multiple signaling pathways are known to be activated by cell swelling and to contribute to cell volume homeostasis. Although cell mechanics and membrane tension have been proposed to couple cell swelling to signaling pathways, the impact of swelling on cellular biomechanics and membrane tension have yet to be fully elucidated. In this study, we use atomic force microscopy under isotonic and hypotonic conditions to measure mechanical properties of endothelial membranes including membrane stiffness, which reflects the stiffness of the submembrane cytoskeleton complex, and the force required for membrane tether formation, reflecting membrane tension and membrane-cytoskeleton attachment. We find that hypotonic swelling results in significant stiffening of the endothelial membrane without a change in membrane tension/membrane-cytoskeleton attachment. Furthermore, depolymerization of F-actin, which, as expected, results in a dramatic decrease in the cellular elastic modulus of both the membrane and the deeper cytoskeleton, indicating a collapse of the cytoskeleton scaffold, does not abrogate swelling-induced stiffening of the membrane. Instead, this swelling-induced stiffening of the membrane is enhanced. We propose that the membrane stiffening should be attributed to an increase in hydrostatic pressure that results from an influx of solutes and water into the cells. Most importantly, our results suggest that increased hydrostatic pressure, rather than changes in membrane tension, could be responsible for activating volume-sensitive mechanisms in hypotonically swollen cells.

Structural perturbation of a dipalmitoylphosphatidylcholine (DPPC) bilayer by warfarin and its bolaamphiphilic analogue: A molecular dynamics study.

Compounds with nominally similar biological activity may exhibit differential toxicity due to differences in their interactions with cell membranes. Many pharmaceutical compounds are amphiphilic and can be taken up by phospholipid bilayers, interacting strongly with the lipid-aqueous interface whether or not subsequent permeation through the bilayer is possible. Bolaamphiphilic compounds, which possess two hydrophilic ends and a hydrophobic linker, can likewise undergo spontaneous uptake by bilayers. While membrane-spanning bolaamphiphiles can stabilize membranes, small molecules with this characteristic have the potential to create membrane defects via disruption of bilayer structure and dynamics. When compared to the amphiphilic therapeutic anticoagulant, warfarin, the bolaamphiphilic analogue, brodifacoum, exhibits heightened toxicity that goes beyond superior inhibition of the pharmacological target enzyme. We explore, herein, the consequences of anticoagulant accumulation in a dipalmitoylphosphatidylcholine (DPPC) bilayer. Coarse-grained molecular dynamics simulations reveal that permeation of phospholipid bilayers by brodifacoum causes a disruption of membrane barrier function that is driven by the bolaamphiphilic nature and size of this molecule. We find that brodifacoum partitioning into bilayers causes membrane thinning and permeabilization and promotes lipid flip-flop - phenomena that are suspected to play a role in triggering cell death. These phenomena are either absent or less pronounced in the case of the less toxic, amphiphilic compound, warfarin.