Dynamic Reorganization and Correlation among Lipid Raft Components.

Lipid rafts are widely believed to be an essential organizational motif in cell membranes. However, direct evidence for interactions among lipid and/or protein components believed to be associated with rafts is quite limited owing, in part, to the small size and intrinsically dynamic interactions that lead to raft formation. Here, we exploit the single negative charge on the monosialoganglioside GM1, commonly associated with rafts, to create a gradient of GM1 in response to an electric field applied parallel to a patterned supported lipid bilayer.

α-Synuclein-induced tubule formation in lipid bilayers.

α-Synuclein is a presynaptic protein that binds to phospholipid membranes and is involved in the pathogenesis of Parkinson's disease (PD). In this paper, we describe the effects of adding wild-type α-synuclein (WT) and three familial PD mutants (A53T, A30P, and E46K) to membranes containing 15-35 mol % anionic lipid. Tubules were observed to form in the membranes to an extent that depended on the α-synuclein variant, the anionic lipid content, and the protein concentration.

Adsorption of alpha-synuclein on lipid bilayers: modulating the structure and stability of protein assemblies.

The interaction of alpha-synuclein with phospholipid membranes has been examined using supported lipid bilayers and epi-fluorescence microscopy. The membranes contained phosphatidylcholine (PC) and phosphatidic acid (PA), which mix at physiological pH. Upon protein adsorption, the lipids undergo fluid-fluid phase separation into PC-rich and PA-rich regions.

Path dependence of three-phase or two-phase end points in fluid binary lipid mixtures.

The phase behavior of anionic/zwitterionic mixtures can be controlled by tuning the charge state of the anionic lipid. In the case of dioleoylphosphatidic acid (DOPA)/dioleoylphosphatidylcholine (DOPC) mixtures, demixing occurs either when DOPA is protonated or when DOPA(2-):Ca(2+) complexes form. Herein it will be shown that the final end point, a three-phase or two-phase system, depends on the order in which the charge state is manipulated.

Clustering of alpha-synuclein on supported lipid bilayers: role of anionic lipid, protein, and divalent ion concentration.

Alpha-synuclein is the major component of Lewy body inclusions found in the brains of patients with Parkinson's disease. Several studies indicate that alpha-synuclein binds to negatively charged phospholipid bilayers. We examined the binding of alpha-synuclein to membranes containing different amounts of negatively charged lipids using supported lipid bilayers, epifluorescence microscopy, fluorescence recovery after photobleaching, and bulk fluorescence techniques.

Formation of complex three-dimensional structures in supported lipid bilayers.

Cell membranes are continually undergoing a wide range of shape transformations. Here, we demonstrate the formation of several structures in supported bilayers, including tubules, caps, and giant multivesicular structures. The key elements required for these transformations are osmotic pressure imbalances, insertion of lipids with positive curvature, and lipids whose curvature is dependent on the screening environment.

Controlling the charge and organization of anionic lipid bilayers: effect of monovalent and divalent ions.

It is shown that the organization of lipid bilayers containing phosphatidic acid (PA) and phosphatidlycholine (PC) can be controlled by altering the monovalent and divalent ion concentrations. At high pH and/or calcium concentration, 1:1 Ca(2+)-PA(2-) complexes form; these complexes demix, and PA-rich and PC-rich regions are observable with epifluorescence microscopy. The results are compared with predictions from electrostatic theory.

Using ionic strength to control liquid-ordered/liquid-disordered separation.

In this Letter, we will show that liquid-ordered/liquid-disordered separation can be controlled with ionic strength. Using this observation, a robust method was developed for creating visible, by fluorescence microscopy, liquid-ordered domains in supported lipid bilayers. The details of the method will be discussed.

Effect of ions on the organization of phosphatidylcholine/phosphatidic acid bilayers.

Lipid bilayers are two-dimensional fluids. Here, the effect of monovalent ion concentration on the mixing, and consequently the organization, of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/1,2-dioleoyl-sn-glycero-3-phosphate (DOPA) bilayers has been examined. Epifluorescence microscopy was used to visualize the organization.

Formation of three-dimensional structures in supported lipid bilayers.

The creation of three-dimensional structures in supported lipid bilayers has been examined. In bilayers, shape transformations can be triggered by adjusting a variety of parameters. Here, it is shown that bilayers composed of phosphatidylcholine and phosphatidic acid can be induced to reversibly form cap structures when exposed to an asymmetry in ionic strength.

Effect of surface treatment on diffusion and domain formation in supported lipid bilayers.

Supported lipid bilayers are widely used as model systems due to their robustness. Due to the solid support, the properties of supported lipid bilayers are different from those of freestanding bilayers. In this article, we examine whether different surface treatments affect the properties of supported lipid bilayers.

Influence of lipid chemistry on membrane fluidity: tail and headgroup interactions.

Membrane fluidity plays an important role in cell function and may, in many instances, be adjusted to facilitate specific cellular processes. To understand better the effect that lipid chemistry has on membrane fluidity the inclusion of three different lipids into egg phosphatidylcholine (eggPC) bilayers has been examined; the three lipids are egg phosphatidylethanolamine ((eggPE) made by transphosphatidylation of eggPC in the presence of ethanolamine), lyso-phosphatidylcholine (LPC), and lyso-phosphatidylethanolamine (LPE). The fluidity of the membranes was determined using fluorescence recovery after photobleaching and the intermolecular interactions were examined using attenuated total reflection Fourier transform infrared spectroscopy.

Effect of salt concentration on membrane lysis pressure.

Cell membranes are capable of withstanding significant osmotic stress, the exact amount of which varies with the lipid composition. In this paper, we examine the effect that salt concentration has on the lysis pressure of membranes containing anionic lipids. Vesicles containing varying amounts of phosphatidylcholine and phosphatidylglycerol were osmotically stressed using NaCl as the osmolyte.

Infrared spectroscopy of fluid lipid bilayers.

Infrared spectroscopy is a powerful technique for examining lipid bilayers; however, it says little about the fluidity of the bilayer-a key physical aspect. It is shown here that it is possible to both acquire spectroscopic data of supported lipid bilayer samples and make measurements of the membrane fluidity. Attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FT-IR) is used to obtain the spectroscopic information and fluorescence recovery after photobleaching (FRAP) is used to determine the fluidity of the samples.