Phase-separated Lipid Bilayers Research Articles

Cholesterol-Dependence of the Rupture Activation Energy in Phase-Segregated Multicomponent Lipid Bilayers

The role of cholesterol in rafts assembly has been demonstrated in both model membranes and living cells. Here, we used AFM-based force mapping to investigate the influence of different cholesterol levels (5-40%) on the rupture of phase-separated multicomponent lipid bilayers consisting of DOPC/sphingomyelin/cholesterol. We report breakthrough forces for the coexisting phases, liquid ordered domains (Lo) and fluid disordered phase (Ld), at various loading rates. Breakthrough forces for both Lo and Ld phases increase with higher loading rate and decrease with increasing cholesterol concentration, consistent with the role of cholesterol in the formation of liquid ordered state. The breakthrough activation energies (30-60 kJ/mol) were calculated following the model for rupture of molecular thin films and compare favourably with reported values for the diffusion of lipid molecules in phosholipid bilayers using other techniques. This work has demonstrated the effective use of AFM-based force mapping to study the influence of cholesterol concentration on the nanomechanical stability of the coexisting phases of model membranes, providing fundamental nanomechanical insights on the role of cholesterol in the formation and stability of sphingolipid/cholesterol-enriched domains or rafts.

Effect of Support Corrugation on Silica Xerogel−Supported Phase-Separated Lipid Bilayers

Lipid bilayers supported by substrates with nanometer-scale surface corrugations hold interest in understanding both nanoparticle-membrane interactions and the challenges of constructing models of cell membranes on surfaces with desirable properties, e.g., porosity. Here, we successfully form a two-phase (gel-fluid) lipid bilayer supported by nanoporous silica xerogel. Surface topology, lateral diffusion coefficient, and lipid density in comparison to mica-supported lipid bilayers were characterized by atomic force microscopy, fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS), and quantitative fluorescence microscopy, respectively. We found that the two-phase lipid bilayer follows the silica xerogel surface contours. The corrugation imparted on the lipid bilayer results in a lipid density that is twice that on a flat mica surface in the fluid regions. In direct agreement with the doubling of actual bilayer area in a projected area, we find that the lateral diffusion coefficient (D) of fluid lipids on silica xerogel (approximately 1.7 microm2/s) is lower than on mica (approximately 3.9 microm2/s) by both FRAP and FCS techniques. Furthermore, the gel-phase domains on silica xerogel compared to mica were larger and less numerous. Overall, our results suggest the presence of a relatively defect-free continuous two-phase lipid bilayer that penetrates approximately midway into the first layer of approximately 50 nm silica xerogel beads.

Phase Separation and Fractal Domain Formation in Phospholipid/Diacetylene-Supported Lipid Bilayers

Phase separation in lipid bilayers is a phenomenon dependent on many environmental parameters such as pH, temperature, ionic strength, and pressure. Its importance in biological systems is reflected by the fact that it has been implicated in the spatial reorganization of plasma membranes, which leads to signaling and stimulation. Here, we present the study of phase separation, domain formation, and domain morphology of supported lipid bilayers composed of mixtures of diacetylene lipids and phospholipids. We have used high-resolution fluorescence and atomic force microscopy to characterize the phase separation between these lipids, and have found that at temperatures below 40 degrees C diacetylene molecules form fractal-like domains. These molecules aggregate in tetralayer stacks with an average monolayer thickness of 3 nm. Boundary and area fractal dimensions were calculated to quantify the domain growth and morphology. A transition from dendritic to dense branching growth was observed as the relative diacetylene concentration was increased. The ability to tailor the growth pattern by changing the relative amount of diacetylene molecules makes this a useful model system for the study of nonequilibrium growth phenomena. In addition, we have explored the possibility of promoting diacetylene domain nucleation through the use of nanostructured surfaces. We found that nanoscale perturbations acted as nucleation sites and modified the growth pattern of diacetylene domains. Phase separation induced by nanometer-scale perturbations could prove useful in selectively positioning lipid patches with specific compositions.

Pore Formation by a Bax-Derived Peptide: Effect on the Line Tension of the Membrane Probed by AFM

Bax is a critical regulator of physiological cell death that increases the permeability of the outer mitochondrial membrane and facilitates the release of the so-called apoptotic factors during apoptosis. The molecular mechanism of action is unknown, but it probably involves the formation of partially lipidic pores induced by Bax. To investigate the interaction of Bax with lipid membranes and the physical changes underlying the formation of Bax pores, we used an active peptide derived from helix 5 of this protein (Bax- α5) that is able to induce Bax-like pores in lipid bilayers. We report the decrease of line tension due to peptide binding both at the domain interface in phase-separated lipid bilayers and at the pore edge in atomic force microscopy film-rupture experiments. Such a decrease in line tension may be a general strategy of pore-forming peptides and proteins, as it affects the energetics of the pore and stabilizes the open state.

Ceramide Promotes Restructuring of Model Raft Membranes

The generation of ceramide in cellular membranes is believed to cause coalescence of small lipid raft domains to give large signaling platforms, thus providing a site for the oligomerization of cell surface receptors. We have used atomic force microscopy to study the effects of ceramide generation by in situ enzymatic hydrolysis of sphingomyelin in phase-separated lipid bilayers that have sphingomyelin/cholesterol-rich domains surrounded by a fluid phase. In situ generation of ceramide produces heterogeneous domains with many raised subdomains that are also formed in bilayers containing premixed ceramide. However, in situ ceramide generation also results in the restructuring of the bilayer to give (1) areas of fluid phase that are devoid of domains, (2) areas that have a distribution of domains similar to the original bilayer, and (3) areas containing clusters of domains. The observation of the ceramide-promoted heterogeneity and clustering of raft domains in a physiologically relevant model provides strong support for the ceramide-induced formation of signaling platforms in cell membranes.

Phase behavior and the partitioning of caveolin-1 scaffolding domain peptides in model lipid bilayers

The membrane binding and model lipid raft interaction of synthetic peptides derived from the caveolin scaffolding domain (CSD) of the protein caveolin-1 have been investigated. CSD peptides bind preferentially to liquid-disordered domains in model lipid bilayers composed of cholesterol and an equimolar ratio of dioleoylphosphatidylcholine (DOPC) and brain sphingomyelin. Three caveolin-1 peptides were studied: the scaffolding domain (residues 83–101), a water-insoluble construct containing residues 89–101, and a water-soluble construct containing residues 89–101. Confocal and fluorescence microscopy investigation shows that the caveolin-1 peptides bind to the more fluid cholesterol-poor phase. The binding of the water-soluble peptide to lipid bilayers was measured using fluorescence correlation spectroscopy (FCS). We measured molar partition coefficients of 10 4 M −1 between the soluble peptide and phase-separated lipid bilayers and 10 3 M −1 between the soluble peptide and bilayers with a single liquid phase. Partial phase diagrams for our phase-separating lipid mixture with added caveolin-1 peptides were measured using fluorescence microscopy. The water-soluble peptide did not change the phase morphology or the miscibility transition in giant unilamellar vesicles (GUVs); however, the water-insoluble and full-length CSD peptides lowered the liquid–liquid melting temperature.

The atomic force microscope as a tool for studying phase separation in lipid membranes (Review)

Atomic force microscopy has developed into a powerful tool in the study of phase separation in lipid bilayers. Its ability to image a semi-fluid surface under buffer at nanometre lateral resolution and Angstrom resolution vertically allows us to distinguish phase separated lipid domains, models of the elusive rafts postulated to exist as functional platforms in the cellular membrane, which may only rise 0.3 nm above the surrounding membrane. This review charts the history of this development, and includes a description of sample preparation techniques, factors affecting image contrast mechanisms, its use in the investigation of the pre-transition ripple phase, and in the localization of cell surface proteins.

Formation and Spatio-Temporal Evolution of Periodic Structures in Lipid Bilayers

Miscibility phase separation in lipid bilayers is widely implicated as an organizing principle in living cell membranes. However, the chemical and physical aspects of how membrane phase separation modulates protein activity remain obscure. Herein, we describe formation of ordered superstructures of coexisting liquid phases in bilayer membranes. Metastable stripe and hexagonal domain lattices are observed, as well as transitions between them. The high degree of order achieved by these methods facilitates statistical analysis of domain spatial distributions and enables measurement of domain interactions. Such long-range ordering principles may exist in more complicated membrane systems.

Scanning force microscopy based rapid force curve acquisition on supported lipid bilayers: experiments and simulations using pulsed force mode.

In situ pulsed force mode scanning force microscopy (PFM-SFM) images of phase separated solid-supported lipid bilayers are discussed with the help of computer simulations. Simultaneous imaging of material properties and topography in a liquid environment by means of PFM-SFM is severely hampered by hydrodynamic damping of the cantilever. Stiffness and adhesion images of solid-supported membranes consisting of cholesterol, sphingomyelin, and 1,2-dioleyl-phosphatidylcholine obtained in aqueous solution exhibit contrast inversion of adhesion and stiff. ness images depending on parameters such as driving frequency, amplitude, and trigger setting. Simulations using a simple harmonic oscillator model explain experimental findings and give a deeper insight into the way PFM-SFM experiments have to be performed in order to obtain interpretable results and hence pave the way for reliable material contrast imaging at high speed.

Population genetic responses to environmental stress in invertebrates

Nonideal mixing provides the physical basis of domain formation and macroscopic phase separation in lipid bilayers. We present a model for the electrostatic contribution to the nonideality of a two-component acidic–zwitterionic lipid membrane. Our model is based on the mean-field Poisson–Boltzmann approach; it includes a protonation/deprotonation equilibrium applicable to acidic lipids such as phosphatidic acid or phosphatidylglycerol. It also includes an electrostatic model for zwitterionic lipids such as phosphatidylcholine or phosphatidylethanolamine that accounts for the spatial separation of the two headgroup charges and the orientational freedom of the headgroup. Modeling the nonelectrostatic contribution to the free energy using the Bragg–Williams approximation of a binary lattice gas enables us to compute binodal lines that reflect the influence of membrane electrostatics on the nonideal mixing properties of the bilayer. If we neglect the zwitterionic lipid's electrostatic contribution, then increasing pH is predicted to always oppose demixing, stabilizing the membrane due to the repulsion between the charged lipids. If the electrostatic properties of the zwitterionic lipids are accounted for, this opposing tendency weakens and may even reverse. In this case, increasing the fraction of charged lipids through increasing pH would, somewhat unexpectedly, promote domain formation. We discuss the corresponding physical mechanism.

Phase separation in lipid bilayers containing integral proteins. Computer simulation studies.

Phase separation in lipid bilayers containing integral proteins. Computer simulation studies.