Planar-supported Bilayers Research Articles

Raman Microscopy Investigation of GLP-1 Peptide Association with Supported Phospholipid Bilayers

A wide range of important biological processes occur at phospholipid membranes including cell signaling, where a peptide or small molecule targets a membrane-localized receptor protein. In this work, we report the adaptation of confocal Raman microscopy to quantify populations of unlabeled glucagon-like peptide-1 (GLP-1), a membrane-active 30-residue incretin peptide, in supported phospholipid bilayers deposited on the interior surfaces of wide-pore porous silica particles. Quantification of lipid bilayer-associated peptide is achieved by measuring the Raman scattering intensity of the peptide relative to that of the supported lipid bilayer, which serves as an internal standard. The dependence of the bilayer-associated GLP-1 population on the solution concentration of GLP-1 produces an isotherm used to determine the equilibrium constant for peptide-bilayer association and the maximum peptide surface coverage. The maximum coverage of GLP-1 in the lipid bilayer was found to be only 1/5th of a full monolayer based on its hydrodynamic radius. The saturation coverage, therefore, is not limited by the size of GLP-1 but by the ability of the bilayer to accommodate the peptide at high concentrations within the bilayer. Raman spectra show that GLP-1 association with the supported bilayer is accompanied by structural changes consistent with the intercalation of the peptide into the bilayer, where the observed increase in acyl-chain order would increase the lipid density and provide free volume needed to accommodate the peptide. These results were compared with previous measurements of the association of fluorescently labeled GLP-1 with a planar-supported bilayer; the unlabeled peptide exhibits a 3-fold greater affinity for the lipid bilayer on the porous silica support, suggesting that the fluorescent label alters the GLP-1 lipid bilayer association.

Asymmetric Phosphatidylethanolamine Distribution Controls Fusion Pore Lifetime and Probability

Little attention has been given to how the asymmetric lipid distribution of the plasma membrane might facilitate fusion pore formation during exocytosis. Phosphatidylethanolamine (PE), a cone-shaped phospholipid, is predominantly located in the inner leaflet of the plasma membrane and has been proposed to promote membrane deformation and stabilize fusion pores during exocytotic events. To explore this possibility, we modeled exocytosis using plasma membrane SNARE-containing planar-supported bilayers and purified neuroendocrine dense core vesicles (DCVs) as fusion partners, and we examined how different PE distributions between the two leaflets of the supported bilayers affected SNARE-mediated fusion. Using total internal reflection fluorescence microscopy, the fusion of single DCVs with the planar-supported bilayer was monitored by observing DCV-associated neuropeptide Y tagged with a fluorescent protein. The time-dependent line shape of the fluorescent signal enables detection of DCV docking, fusion-pore opening, and vesicle collapse into the planar membrane. Four different distributions of PE in the planar bilayer mimicking the plasma membrane were examined: exclusively in the leaflet facing the DCVs; exclusively in the opposite leaflet; equally distributed in both leaflets; and absent from both leaflets. With PE in the leaflet facing the DCVs, overall fusion was most efficient and the extended fusion pore lifetime (0.7 s) enabled notable detection of content release preceding vesicle collapse. All other PE distributions decreased fusion efficiency, altered pore lifetime, and reduced content release. With PE exclusively in the opposite leaflet, resolution of pore opening and content release was lost.

Assembly and Comparison of Plasma Membrane SNARE Acceptor Complexes

Neuronal exocytotic membrane fusion occurs on a fast timescale and is dependent on interactions between the vesicle SNARE synaptobrevin-2 and the plasma membrane SNAREs syntaxin-1a and SNAP-25 with a 1:1:1 stoichiometry. Reproducing fast fusion rates as observed in cells by reconstitution in vitro has been hindered by the spontaneous assembly of a 2:1 syntaxin-1a:SNAP-25 complex on target membranes that kinetically alters the binding of synaptobrevin-2. Previously, an artificial SNARE acceptor complex consisting of 1:1:1 syntaxin-1a(residues 183–288):SNAP-25:syb(residues 49–96) was found to greatly accelerate the rates of lipid mixing of reconstituted target and vesicle SNARE proteoliposomes. Here we present two (to our knowledge) new procedures to assemble membrane-bound 1:1 SNARE acceptor complexes that produce fast and efficient fusion without the need of the syb(49–96) peptide. In the first procedure, syntaxin-1a is purified in a strictly monomeric form and subsequently assembled with SNAP-25 in detergent with the correct 1:1 stoichiometry. In the second procedure, monomeric syntaxin-1a and dodecylated (d-)SNAP-25 are separately reconstituted into proteoliposomes and subsequently assembled in the plane of merged target lipid bilayers. Examining single particle fusion between synaptobrevin-2 proteoliposomes and planar-supported bilayers containing the two different SNARE acceptor complexes revealed similar fast rates of fusion. Changing the stoichiometry of syntaxin-1a and d-SNAP-25 in the target bilayer had significant effects on docking, but little effect on the rates of synaptobrevin-2 proteoliposome fusion.

Single Vesicle Millisecond Fusion Kinetics Reveals Number of SNARE Complexes Optimal for Fast SNARE-mediated Membrane Fusion

SNAREs mediate membrane fusion in intracellular vesicle traffic and neuronal exocytosis. Reconstitution of membrane fusion in vitro proved that SNAREs constitute the minimal fusion machinery. However, the slow fusion rates observed in these systems are incompatible with those required in neurotransmission. Here we present a single vesicle fusion assay that records individual SNARE-mediated fusion events with millisecond time resolution. Docking and fusion of reconstituted synaptobrevin vesicles to target SNARE complex-containing planar membranes are distinguished by total internal reflection fluorescence microscopy as separate events. Docking and fusion are SNAP-25-dependent, require no Ca(2+), and are efficient at room temperature. Analysis of the stochastic data with sequential and parallel multi-particle activation models reveals six to nine fast-activating steps. Of all the tested models, the kinetic model consisting of eight parallel reaction rates statistically fits the data best. This might be interpreted by fusion sites consisting of eight SNARE complexes that each activate in a single rate-limiting step in 8 ms.

Transbilayer Effects of Raft-Like Lipid Domains in Asymmetric Planar Bilayers Measured by Single Molecule Tracking

Cell membranes have complex lipid compositions, including an asymmetric distribution of phospholipids between the opposing leaflets of the bilayer. Although it has been demonstrated that the lipid composition of the outer leaflet of the plasma membrane is sufficient for the formation of raft-like liquid-ordered ( l o) phase domains, the influence that such domains may have on the lipids and proteins of the inner leaflet remains unknown. We used tethered polymer supports and a combined Langmuir-Blodgett/vesicle fusion (LB/VF) technique to build asymmetric planar bilayers that mimic plasma membrane asymmetry in many ways. We show that directly supported LB monolayers containing cholesterol-rich l o phases are inherently unstable when exposed to water or vesicle suspensions. However, tethering the LB monolayer to the solid support with the lipid-anchored polymer 1,2-dimyristoyl phophatidylethanolamine- N-[poly(ethylene glycol)-triethoxysilane] significantly improves stability and allows for the formation of complex planar-supported bilayers that retain >90% asymmetry for 1–2 h. We developed a single molecule tracking (SPT) system for the study of lipid diffusion in asymmetric bilayers with coexisting liquid phases. SPT allowed us to study in detail the diffusion of individual lipids inside, outside, or directly opposed to l o phase domains. We show here that l o phase domains in one monolayer of an asymmetric bilayer do not induce the formation of domains in the opposite leaflet when this leaflet is composed of palmitoyl-oleoyl phosphatidylcholine and cholesterol but do induce domains when this leaflet is composed of porcine brain phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and cholesterol. The diffusion of lipids is similar in l o and liquid-disordered phase domains and is not affected by transbilayer coupling, indicating that lateral and transverse lipid interactions that give rise to the domain structure are weak in the biological lipid mixtures that were employed in this work.

Imaging Fast SNARE Mediated-Membrane Fusion in Planar-Supported Bilayers

Seven years ago, Rothman and co-workers reported that the pairing of SNARE molecules constitutes a minimal machinery for the fusion of two membranes (1), providing support for the earlier formulated SNARE hypothesis. Their conclusion was drawn from experiments in vitro in which the plasma membrane protein complex syntaxin1A/SNAP25 as t-SNARE and the synaptic vesicle membrane protein vamp2 as v-SNARE were reconstituted into lipid vesicles. Although this assay clearly demonstrated that t- and v-SNAREs were sufficient to fuse liposomes, the kinetics of the reaction were disturbingly slow. The half-time of fusion was on the order of 10 min, i.e., >105 times slower than Ca2+-triggered synaptic vesicle fusion in neurons (2).

Electric field-induced critical demixing in lipid bilayer membranes

Electric fields can induce lateral reorganization of lipids in fluid bilayer membranes. The resulting concentration profiles readily are observed in planar-supported bilayers by epifluorescence microscopy. When a fluorescently labeled lipid was used to probe the field-induced separation of cardiolipin from egg-phosphatidylcholine, an enhanced sensitivity to the electric field was observed that is attributed to a critical demixing effect. A thermodynamic model of the system was used to analyze the results. The observed concentration profiles can be understood if the lipid mixture has a critical temperature equal to 75 degrees K. The steady-state distribution of lipids under the influence of an electric field is very sensitive to demixing effects, even at temperatures well above the critical temperature for spontaneous phase separation, and this may have significant consequences for organization and structural changes in natural cell membranes.