Juan M. Vanegas

AFM for structure and dynamics of biomembranes.

We review structure and dynamic measurements of biomembranes by atomic force microscopy (AFM). We focus mainly on studies involving supported lipid bilayers (SLBs), particularly formation by vesicle rupture on flat and corrugated surfaces, nucleation and growth of domains in phase-separated systems, anesthetic-lipid interactions, and protein/peptide interactions in multicomponent systems. We show that carefully designed experiments along with real-time AFM imaging with superior lateral and z resolution (0.1 nm) have revealed quantitative details of the mechanisms and factors controlling vesicle rupture, domain shape and size, phase transformations, and some model biological interactions. The AFM tip can also be used as a mechanical transducer and incorporated in electrochemical measurements of membrane components; therefore, we touch on these important applications in both model and cell membranes.

Role of Unsaturated Lipid and Ergosterol in Ethanol Tolerance of Model Yeast Biomembranes

We present a combined atomic force microscopy and fluorescence microscopy study of the behavior of a ternary supported lipid bilayer system containing a saturated lipid (DPPC), an unsaturated lipid (DOPC), and ergosterol in the presence of high ethanol (20 vol %). We find that the fluorescent probe Texas Red DHPE preferentially partitions into the ethanol-induced interdigitated phase, which allows the use of fluorescence imaging to investigate the phase behavior of the system. Atomic force microscopy and fluorescence images of samples with the same lipid mixture show good agreement in sample morphology and area fractions of the observed phases. Using area fractions obtained from fluorescence images over a broad range of compositions, we constructed a phase diagram of the DPPC/DOPC/ergosterol system at 20 vol % ethanol. The phase diagram clearly shows that increasing unsaturated lipid and/or ergosterol protects the membrane by preventing the formation of the interdigitated phase. This result supports the hypothesis that yeast cells increase ergosterol and unsaturated lipid content to prevent interdigitation and maintain an optimal membrane thickness as ethanol concentration increases during anaerobic fermentations. Changes in plasma membrane composition provide an important survival factor for yeast cells to deter ethanol toxicity.

Crystalline, Ordered and Disordered Lipid Membranes: Convergence of Stress Profiles due to Ergosterol

We present a simulation study focusing on modulations of the stress, or lateral pressure, profiles of lipid bilayer phases by addition of a sterol, ergosterol, at multiple temperatures. A major redistribution of lateral and normal pressures across the gel-phase bilayer required 10 mol % sterol in comparison to a gradual redistribution beginning at 20 mol % for the liquid phase. Stress profiles across all temperatures converged at 30 mol % ergosterol. Redistribution and merging of stress profiles, associated with structural alterations, are coincident with experimentally observed modulations in mechanical properties and therefore are suggested as the mechanism of action for this biologically necessary role of sterols.