In vivo lipid membranes interact with rough supramolecular structures such as protein clusters and fibrils. How these features whose size ranges from a few nanometers to a few tens of nanometers impact lipid and protein mobility is still being investigated. Here, we study supported phospholipid bilayers, a unique biomimetic model, deposited on etched surfaces bearing nanometric corrugations. The surface roughness and mean curvature are carefully characterized by AFM imaging using ultrasharp tips. Neutron specular reflectivity supplements this surface characterization and indicates that the bilayers follow the large-scale corrugations of the substrate. We measure the lateral mobility of lipids in both the fluid and gel phases by fluorescence recovery after patterned photobleaching. Although the mobility is independent of the roughness in the gel phase, it exhibits a 5-fold decrease in the fluid phase when the roughness increases from 0.2 to 10 nm. These results are interpreted with a two-phase model allowing for a strong decrease in the lipid mobility in highly curved or defect-induced gel-like nanoscale regions. This suggests a strong link between membrane curvature and fluidity, which is a key property for various cell functions such as signaling and adhesion.
To determine how lipid bilayer/support interactions are affected by ionic strength, we carried out lipid diffusion coefficient measurements by fluorescence recovery after patterned photobleaching (FRAPP) and transfer ratio measurements using a Langmuir balance on supported bilayers of phosphatidylcholine lipids. The main effect of increasing ionic strength is shown to be enhanced diffusion of the lipids due to a decrease in the electrostatic interaction between the bilayer and the support. We experimentally confirm that the two main parameters governing bilayer behavior are electrostatic interaction and bilayer/support distance. Both these parameters can therefore be used to vary the potential that acts on the bilayer. Additionally, our findings show that FRAPP is an extremely sensitive tool to study interaction effects: here, variations in diffusion coefficient as well as the presence or absence of leaflet decoupling.
Cell mechanisms are actively modulated by membrane dynamics. We studied the dynamics of a first-stage biomimetic system by Fluorescence Recovery After Patterned Photobleaching. Using this simple biomimetic system, constituted by α -hemolysin from Staphylococcus aureus inserted as single heptameric pore or complexes of pores in a glass-supported DMPC bilayer, we observed true diffusion behavior, with no immobile fraction. We find two situations: i) when incubation is shorter than 15 hours, the protein inserts as a heptameric pore and diffuses roughly three times more slowly than its host lipid bilayer; ii) incubation longer than 15 hours leads to the formation of larger complexes which diffuse more slowly. Our results indicate that, while the Saffman-Delbruck model adequately describes the diffusion coefficient D for small radii, D of the objects decreases as 1/R(2) for the size range explored in this study. Additionally, in the presence of inserted proteins, the gel-to-fluid transition of the supported bilayer as well as a temperature shift in the gel-to-fluid transition are observed.
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