Ceramide and Cholesterol Effects on Phospholipid Bilayers under the AFM: Characterization of Complex Lipid Phases

Abstract

Atomic force microscopy (AFM) has been applied to the characterization of palmitoylceramide (pCer) and cholesterol (Chol) incorporation into phospholipid-based supported planar bilayers (SPBs) at 22°C. Phospholipids were dipalmitoyl phosphatidylcholine (DPPC) or palmitoyl sphingomyelin (pSM). Membranes of different compositions were prepared by either the vesicle adsorption or the spin-coating procedures (the latter strictly for pCer-containing mixtures) and analyzed with a combination of AFM imaging (domain segregation, bilayer thickness and roughness) and force spectroscopy (mechanical resistance to indentation). The mixtures under study were pure phospholipids (pSM, DPPC), phospholipid:Chol (70:30), phospholipid:Chol:pCer (2:1:1) and phospholipid:pCer (90:10, 80:20 and 70:30). Binary phospholipid:pCer mixtures at increasing ceramide ratios gave rise to highly-resistant segregated domains with increasing extension but similar properties in terms of breakthrough forces, thicknesses and roughnesses. These ceramide-enriched domains are able to exclude a fluorescent lipid probe (DiIC18) due to their high intermolecular packing. Interestingly, these domains have been reported to disappear when model membranes become highly enriched in cholesterol in fluid membranes or in the absence of a fluid phase (our case). Indeed, the ternary mixtures (2:1:1) gave rise to a homogenous lamellar gel phase with significantly different properties when compared to all of the other mixtures under study: ternary mixtures showed a reduced thickness and an intermediate roughness and mechanical resistance when compared to phospholipid:Chol (70:30) and phospholipid:pCer. These differences were statistically significant. More importantly, at those relatively high pCer and Chol concentrations in ternary mixtures, no mutual displacement of these molecules was observed. The data become relevant in the context of sphingolipid signaling and membrane platform formation.