Crystalline hydration structure at the membrane-fluid interface of model lipid rafts indicates a highly reactive boundary region.

The fluid mosaic model of biological membranes is that of a two-dimensional lipid bilayer in which both lipids and associated membrane proteins diffuse freely. More recently, the raft hypothesis proposed that membranes contain small, dynamic, functional domains (rafts), which act as platforms for membrane protein attachment and interaction. Although experimental evidence supporting the raft hypothesis is growing, very little is known of the structure of the membrane-fluid interface of lipid raft systems. Here, we report the direct submolecular-scale imaging of model raft membranes using ultrahigh resolution atomic force microscopy. We characterize the heterogeneous nature of crystalline hydration layers at the membrane-fluid interface. The association of crystalline hydration layers with raft membranes would significantly affect the mechanism and kinetics of both inter-raft interactions and those between rafts and external biomolecules, and therefore this finding has important implications for membrane biology.

Direct submolecular scale imaging of mesoscale molecular order in supported dipalmitoylphosphatidylcholine bilayers.

Supported dipalmitoylphosphatidylcholine (DPPC) bilayers are widely used membrane systems in biophysical and biochemical studies. Previously, short-range positional and orientational order of lipid headgroups of supported DPPC bilayers was observed at room temperature using low deflection noise frequency modulation atomic force microscopy (FM-AFM). While this ordering was supported by X-ray diffraction studies, it conflicted with diffusion coefficient measurements of gel-phase bilayers determined from fluorescence photobleaching experiments. In this work, we have directly imaged mica-supported DPPC bilayers with submolecular resolution over scan ranges up to 146 nm using low deflection noise FM-AFM. Both orientational and positional molecular ordering were observed in the mesoscale, indicative of crystalline order. We discuss these results in relation to previous biophysical studies and propose that the mica support induces mesoscopic crystalline order of the DPPC bilayer at room temperature. This study also demonstrates the recent advance in the scan range of submolecular scale AFM imaging.

A model system to study the insertion of cholesterol into a phospholipid monolayer.

Colloidal probe atomic force microscopy (AFM) was used to study the interaction between a surface bearing tethered cholesterol groups and an egg phosphatidylcholine (egg-PC) monolayer. The cholesterol bearing surface was comprised of a mixed self-assembled monolayer comprised of O-cholesteryl N-(8'-mecapto-3',6'-dioxaoctyl)carbamate (CPEO3) molecules and beta-mercaptoethanol formed on a 20 mum diameter gold-coated silica particle. The egg-PC monolayer was adsorbed onto an octadecylthiol monolayer formed on template-stripped gold. The force between the surfaces, as a function of separation, was measured for surface concentrations of CPEO3 from 0 to 100 mol %. At all concentrations there was a long-range repulsive double-layer force due to weak surface charges. At surface concentrations of CPEO3 from 1 to 29 mol % the interaction on the approach of the surfaces showed a maximum in the repulsive force, followed by a small (2-5 nm) jump into a force minimum corresponding to adhesion of the surfaces. On separation, a normalized pull-off force of 1.0-1.6 mN m(-1) was measured. Over the same concentration range, the calculated interaction energy per CPEO3 molecule decreased from 1.1 +/- 0.2 kT to 0.04 kT. At surface concentrations of 35 mol % and above there was no reproducible adhesion between the cholesterol-bearing surface and the phospholipid monolayer. We attribute the occurrence of short-range attraction and adhesion in the 1-29 mol % regime to the insertion of (some) cholesterol groups into the phospholipid monolayer. At higher surface concentrations the efficiency of insertion is reduced due to steric effects. We discuss the experimental results in the light of the energetics of the insertion of a cholesterol molecule into a lipid bilayer.

Nanoscale mechanical probing of supported lipid bilayers with atomic force microscopy.

We present theory and experiments for the force-distance curve F(z(0)) of an atomic force microscope (AFM) tip (radius R) indenting a supported fluid bilayer (thickness 2d). For realistic conditions the force is dominated by the area compressibility modulus κ(A) of the bilayer and, to an excellent approximation, given by F=πκ(A)Rz(0)(2)/(2d-z(0))(2). The experimental AFM force curves from coexisting liquid ordered and liquid disordered domains in three-component lipid bilayers are well described by our model, which provides κ(A) in agreement with literature values. The liquid ordered phase has a yieldlike response that we model as due to the breaking of hydrogen bonds.

Minimal F-actin cytoskeletal system for planar supported phospholipid bilayers.

Preferential binding of F-actin to lipid bilayers containing ponticulin was investigated on both planar supported bilayers and on a cholesterol-based tethering system. The transmembrane protein ponticulin in Dictyostelium discoideum is known to provide a direct link between the actin cytoskeleton and the cell membrane ( Wuestehube, L. J. ; Luna, E. J. J. Cell Biol. 1987, 105, 1741- 1751 ). Purification of ponticulin has allowed an in vitro model of the F-actin cytoskeletal scaffold system to be formed and investigated by AFM, epi-fluorescence microscopy, surface plasmon resonance (SPR), and quartz crystal microbalance with dissipation (QCM-D). Single filament features of F-actin bound to the ponticulin containing lipid bilayer are shown by AFM to have a pitch of 37.3 +/- 1.1 nm and a filament height of 7.0 +/- 1.6 nm. The complementary techniques of QCM-D and SPR were used to obtain dissociation constants for the interaction of F-actin with ponticulin containing bilayers, giving 10.5 +/- 1.7 microM for a physisorbed bilayer and 10.8 +/- 3.6 microM for a tethered bilayer, respectively.

Titration of ionizable monolayers by measurement of the electric double-layer force.

We have determined the pKa of surface-bound primary amine groups by determining the surface potential as a function of solution pH from the magnitude of the electric double-layer force. Using colloid-probe atomic force microscopy (AFM), we measured the force as a function of separation between a particle of radius R = 10 microm and a planar surface, each coated with a self-assembled monolayer of HS(CH2)2CONH((CH2)2O)8(CH2)2NH2. The force was measured from pH 3 to 7, and the surface potential was determined by fitting the results to solutions of the nonlinear Poisson-Boltzmann equation. The surface pKa of the primary amine group was found to be 5.0 +/- 0.2, in agreement with the results of contact-angle and chemical-force titrations on similar surfaces with primary amine groups. The surface charge density indicates that less than 1% of the NH2 groups are dissociated at pH 3, suggesting that ionization is very unfavorable in the local environment of the ethylene oxide chains. This noncontact method should be of general applicability to surfaces with ionizable groups and avoids the possible complications of large contact forces on the surfaces under study.