Giant Plasma Membrane Vesicles Research Articles

Extracellular domain 2 of TSPAN4 governs its functions

Tetraspanin 4, a protein with four transmembrane helices and three connecting loops, senses membrane curvature and localizes to membrane tubes. This enrichment in tubular membranes enhances its diverse interactions. While the transmembrane part of the protein likely contributes to curvature sensitivity, the possible roles of the ectodomains in curvature sensitivity of tetraspanin 4 are still unknown. Here, using micropipette aspiration combined with confocal microscopy and optical tweezers, we show that the extracellular loop 2 contributes to the curvature sensitivity and curvature-induced interactions of tetraspanin 4. To this end, we created truncated tetraspanin 4 mutants by deleting each of the connecting loops. Subsequently, we pulled membrane tubes from giant plasma membrane vesicles containing tetraspanin 4-GFP or its mutants while maintaining controllable membrane tension and curvature. Among the mutations tested, the removal of the extracellular loop 2 had the most significant impact on both the curvature sensitivity and interactions of tetraspanin 4. Based on the results, we suggest that the extracellular loop 2 regulates the affinity of tetraspanin 4 towards curved membranes and affects its lateral interactions.

Cytotoxic rhamnolipid micelles drive acute virulence in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic human pathogen that has developed multi- or even pan-drug resistance toward most frontline and last resort antibiotics, leading to increasing frequency of infections and deaths among hospitalized patients, especially those with compromised immune systems. Further complicating treatment, P. aeruginosa produces numerous virulence factors that contribute to host tissue damage and immune evasion, promoting bacterial colonization and pathogenesis. In this study, we demonstrate the importance of rhamnolipid production in host-pathogen interactions. Secreted rhamnolipids form micelles that exhibited highly acute toxicity toward murine macrophages, rupturing the plasma membrane and causing organellar membrane damage within minutes of exposure. While rhamnolipid micelles (RMs) were particularly toxic to macrophages, they also caused membrane damage in human lung epithelial cells, red blood cells, Gram-positive bacteria, and even noncellular models like giant plasma membrane vesicles. Most importantly, rhamnolipid production strongly correlated with P. aeruginosa virulence against murine macrophages in various panels of clinical isolates. Altogether, our findings suggest that rhamnolipid micelles are highly cytotoxic virulence factors that drive acute cellular damage and immune evasion during P. aeruginosa infections.

Cell Line and Media Composition Influence the Production of Giant Plasma Membrane Vesicles.

Giant plasma membrane vesicles (GPMVs) have been utilized as a model to study phase separation in the plasma membrane. Additionally, GPMVs have been employed as vehicle for delivering molecular cargo, including small molecule drugs and nanoparticles. Nearly all examples of GPMV production use a defined salt buffer that is a stark contrast to typical cell culture medium. In this study, we demonstrate that the addition of formaldehyde and dithiothreitol to a standard culture medium was capable of generating GPMVs at a concentration equal to or higher than the traditional production buffer. These methods were evaluated for two human cell lines: kidney endothelial and Schwann cells (SCs). Morphological properties of the resultant GPMVs exhibited no significant differences between the two formulations. Factors such as pH and seeding density significantly influenced the production of GPMVs in both mediums. The cell type and seeding density was shown to influence the number of GPMVs to the greatest extent. SCs yield more GPMVs at higher seeding densities compared to endothelial cells. Stability of the membrane of the GPMVs produced in both mediums was evaluated by monitoring passive diffusion of two fluorescently tagged dextrans (3 and 10 kDa). Regardless of the production formulation or cell type, approximately 85% GPMVs are impermeable to either dextran. Cold storage for on-demand use and shipping are essential for broader use of GPMVs. Toward this aim, we have evaluated the GMPV number and morphologies following storage at -80 °C and in liquid nitrogen. A significant loss of the GPMV number, ∼30%, was observed following storage across production formulations as well as cell types. Our results indicate that smaller GMPVs, <5 μm are more stable for preservation. In conclusion, GPMVs can be produced in a broad range of formulations, exhibit a high degree of stability, and can undergo cold storage for further adoption.

Curcumin Modulates the Membrane Raft Integrity via Phase Separation and Induces CD44 Shedding in Tumor Cells.

CD44 is a transmembrane cell adhesion molecule that is cleaved by the membrane proteinase, a disintegrin and metalloproteinase 10 (ADAM10), on the cell surface via ectodomain shedding after cholesterol depletion. Lipid raft-mediated CD44 shedding is essential for cancer cell invasion. As cell-cell and cell-matrix adhesions are critical for cancer progression, lipid raft-targeting agents may be effective for cancer therapy. Here, we found that curcumin and its derivatives induced the ADAM10-mediated shedding of CD44 in tumor cells. We also found that curcumin and the derivatives are membrane-active compounds whose effect depends on its planar backbone and the spatial arrangement of methoxy groups substituted on the two aromatic rings using giant unilamellar and plasma membrane vesicles. Curcumin and its derivatives with rigid backbones and hydroxy groups exerted membrane-domain-modulating activity, which may account for their pleiotropic effects via multiple signaling pathways involving membrane receptors. This study provides a basis for the use of membrane-active compounds, such as curcuminoids, to elucidate the roles of lipid rafts in cellular signaling, regulation of membrane-bound ADAM metalloproteinases, and the development of novel membrane lipid-based therapies.

Lipid Peroxidation Drives Liquid-Liquid Phase Separation and Disrupts Raft Protein Partitioning in Biological Membranes.

The peroxidation of membrane lipids by free radicals contributes to aging, numerous diseases, and ferroptosis, an iron-dependent form of cell death. Peroxidation changes the structure and physicochemical properties of lipids, leading to bilayer thinning, altered fluidity, and increased permeability of membranes in model systems. Whether and how lipid peroxidation impacts the lateral organization of proteins and lipids in biological membranes, however, remains poorly understood. Here, we employ cell-derived giant plasma membrane vesicles (GPMVs) as a model to investigate the impact of lipid peroxidation on ordered membrane domains, often termed membrane rafts. We show that lipid peroxidation induced by the Fenton reaction dramatically enhances the phase separation propensity of GPMVs into coexisting liquid-ordered (Lo) and liquid-disordered (Ld) domains and increases the relative abundance of the disordered phase. Peroxidation also leads to preferential accumulation of peroxidized lipids and 4-hydroxynonenal (4-HNE) adducts in the disordered phase, decreased lipid packing in both Lo and Ld domains, and translocation of multiple classes of raft proteins out of ordered domains. These findings indicate that the peroxidation of plasma membrane lipids disturbs many aspects of membrane rafts, including their stability, abundance, packing, and protein and lipid composition. We propose that these disruptions contribute to the pathological consequences of lipid peroxidation during aging and disease and thus serve as potential targets for therapeutic intervention.

Membrane-Mediated Cooperative Interactions of CD47 and SIRPα.

The specific binding of the ubiquitous 'marker of self' protein CD47 to the SIRPα protein anchored in the macrophage plasma membrane results in the inhibition of the engulfment of 'self' cells by macrophages and thus constitutes a key checkpoint of our innate immune system. Consequently, the CD47-SIRPα protein complex has been recognized as a potential therapeutic target in cancer and inflammation. Here, we introduce a lattice-based mesoscale model for the biomimetic system studied recently in fluorescence microscopy experiments where GFP-tagged CD47 proteins on giant plasma membrane vesicles bind to SIRPα proteins immobilized on a surface. Computer simulations of the lattice-based mesoscale model allow us to study the biomimetic system on multiple length scales, ranging from single nanometers to several micrometers and simultaneously keep track of single CD47-SIRPα binding and unbinding events. Our simulations not only reproduce data from the fluorescence microscopy experiments but also are consistent with results of several other experiments, which validates our numerical approach. In addition, our simulations yield quantitative predictions on the magnitude and range of effective, membrane-mediated attraction between CD47-SIRPα complexes. Such detailed information on CD47-SIRPα interactions cannot be obtained currently from experiments alone. Our simulation results thus extend the present understanding of cooperative effects in CD47-SIRPα interactions and may have an influence on the advancement of new cancer treatments.

EFR3A: a new raft domain organizing protein?

BackgroundMembrane rafts play a crucial role in the regulation of many important biological processes. Our previous data suggest that specific interactions of flotillins with MPP1 are responsible for membrane raft domain organization and regulation in erythroid cells. Interaction of the flotillin-based protein network with specific membrane components underlies the mechanism of raft domain formation and regulation, including in cells with low expression of MPP1.MethodsWe sought to identify other flotillin partners via the immobilized recombinant flotillin-2-based affinity approach and mass spectrometry technique. The results were further confirmed via immunoblotting and via co-immunoprecipitation. In order to study the effect of the candidate protein on the physicochemical properties of the plasma membrane, the gene was knocked down via siRNA, and fluorescence lifetime imaging microscopy and spot-variation fluorescence correlation spectroscopy was employed.ResultsEFR3A was identified as a candidate protein that interacts with flotillin-2. Moreover, this newly discovered interaction was demonstrated via overlay assay using recombinant EFR3A and flotillin-2. EFR3A is a stable component of the detergent-resistant membrane fraction of HeLa cells, and its presence was sensitive to the removal of cholesterol. While silencing the EFR3A gene, we observed decreased order of the plasma membrane of living cells or giant plasma membrane vesicles derived from knocked down cells and altered mobility of the raft probe, as indicated via fluorescence lifetime imaging microscopy and spot-variation fluorescence correlation spectroscopy. Moreover, silencing of EFR3A expression was found to disturb epidermal growth factor receptor and phospholipase C gamma phosphorylation and affect epidermal growth factor-dependent cytosolic Ca2+ concentration.ConclusionsAltogether, our results suggest hitherto unreported flotillin-2-EFR3A interaction, which might be responsible for membrane raft organization and regulation. This implies participation of this interaction in the regulation of multiple cellular processes, including those connected with cell signaling which points to the possible role in human health, in particular human cancer biology.

Giant Plasma Membrane Vesicles as Cellular‐Mimics for Probing SARS‐CoV‐2 Binding at Single Particle Level

AbstractMembrane receptors, attachment factors and various other membrane factors play a critical role in the specific attachment of virus particles to a cell surface. Researchers have been employing a range of model systems starting from receptor‐reconstituted lipid membranes to live cells for quantitative evaluation of virus‐receptor/attachment factor interactions. In this work, we show that giant plasma membrane vesicles (GPMVs) derived from adherent cells are a suitable cell‐mimicking model system to examine the binding of SARS‐CoV‐2 to cellular receptors. We evaluated the binding of cell‐cultured SARS‐CoV‐2 to GPMVs by imaging single virus particles with the wide‐field fluorescence microscopy technique. The GPMVs were derived from three different cell lines, i. e., Vero, HEK293T and A549. We compared the number of bound virus particles per cell, cell‐attached GPMV, and cell‐free GPMV of three different cell lines. We explain the observed trend in the data from the expression level of endogenous ACE2 protein and also, conclude that the protein is functional in GPMVs for virus and antibody binding.

Development of an Effective Functional Lipid Anchor for Membranes (FLAME) for the Bioorthogonal Modification of the Lipid Bilayer of Mesenchymal Stromal Cells.

The glycosylation of cellular membranes is crucial for the survival and communication of cells. As our target is the engineering of the glycocalyx, we designed a functionalized lipid anchor for the introduction into cellular membranes called Functional Lipid Anchor for MEmbranes (FLAME). Since cholesterol incorporates very effectively into membranes, we developed a twice cholesterol-substituted anchor in a total synthesis by applying protecting group chemistry. We labeled the compound with a fluorescent dye, which allows cell visualization. FLAME was successfully incorporated in the membranes of living human mesenchymal stromal cells (hMSC), acting as a temporary, nontoxic marker. The availability of an azido function─a bioorthogonal reacting group within the compound─enables the convenient coupling of alkyne-functionalized molecules, such as fluorophores or saccharides. After the incorporation of FLAME into the plasma membrane of living hMSC, we were able to successfully couple our molecule with an alkyne-tagged fluorophore via click reaction. This suggests that FLAME is useful for the modification of the membrane surface. Coupling FLAME with a galactosamine derivative yielded FLAME-GalNAc, which was incorporated into U2OS cells as well as in giant unilamellar vesicles (GUVs) and cell-derived giant plasma membrane vesicles (GPMVs). With this, we have shown that FLAME-GalNAc is a useful tool for studying the partitioning in the liquid-ordered (Lo) and the liquid-disordered (Ld) phases. The molecular tool can also be used to analyze the diffusion behavior in the model and the cell membranes by fluorescence correlation spectroscopy (FCS).

High efficiency preparation of monodisperse plasma membrane derived extracellular vesicles for therapeutic applications

Extracellular vesicles (EVs) are highly interesting for the design of next-generation therapeutics. However, their preparation methods face challenges in standardization, yield, and reproducibility. Here, we describe a highly efficient and reproducible EV preparation method for monodisperse nano plasma membrane vesicles (nPMVs), which yields 10 to 100 times more particles per cell and hour than conventional EV preparation methods. nPMVs are produced by homogenizing giant plasma membrane vesicles following cell membrane blebbing and apoptotic body secretion induced by chemical stressors. nPMVs showed no significant differences compared to native EVs from the same cell line in cryo-TEM analysis, in vitro cellular interactions, and in vivo biodistribution studies in zebrafish larvae. Proteomics and lipidomics, on the other hand, suggested substantial differences consistent with the divergent origin of these two EV types and indicated that nPMVs primarily derive from apoptotic extracellular vesicles. nPMVs may provide an attractive source for developing EV-based pharmaceutical therapeutics.

Transportan 10 Induces Perturbation and Pores Formation in Giant Plasma Membrane Vesicles Derived from Cancer Liver Cells.

Continuous progress has been made in the development of new molecules for therapeutic purposes. This is driven by the need to address several challenges such as molecular instability and biocompatibility, difficulties in crossing the plasma membrane, and the development of host resistance. In this context, cell-penetrating peptides (CPPs) constitute a promising tool for the development of new therapies due to their intrinsic ability to deliver therapeutic molecules to cells and tissues. These short peptides have gained increasing attention for applications in drug delivery as well as for their antimicrobial and anticancer activity but the general rules regulating the events involved in cellular uptake and in the following processes are still unclear. Here, we use fluorescence microscopy methods to analyze the interactions between the multifunctional peptide Transportan 10 (TP10) and the giant plasma membrane vesicles (GPMVs) derived from cancer cells. This aims to highlight the molecular mechanisms underlying functional interactions which bring its translocation across the membrane or cytotoxic mechanisms leading to membrane collapse and disruption. The Fluorescence Lifetime Imaging Microscopy (FLIM) method coupled with the phasor approach analysis proved to be the winning choice for following highly dynamic spatially heterogeneous events in real-time and highlighting aspects of such complex phenomena. Thanks to the presented approach, we were able to identify and monitor TP10 translocation into the lumen, internalization, and membrane-induced modifications depending on the peptide concentration regime.

High-throughput screening to identify modulators of membrane protein raft affinity in giant plasma membrane vesicles.

Lipid rafts remain an active area of membrane biophysics research. Due to their diffraction-limited size and transient nature direct studies of rafts and their resident proteins in cells remains intractable. The lack of tools to manipulate raft-resident proteins has also stymied our ability to study raft function and raft partitioning of proteins. Giant plasma membrane vesicles (GPMVs), which spontaneously separate into ordered (raft) and disordered (non-raft) phases, are a practical a tool for studying rafts in biological membranes. We recently developed a high-throughput screening pipeline combining high-content imaging with a custom image-analysis software package, VesA, to identify small molecule modulators of phase separation in GPMVs. Using this approach, we are conducting an on-going screen 20,000 of small molecules from the Vanderbilt University Discover collection to find compounds that alter the phase partitioning of the peripheral myelin protein 22 (PMP22). PMP22, a major component of myelin in Schwann cells of the peripheral nervous system, shows a high affinity for ordered phases of GPMVs. Disease causing mutations in PMP22 exhibit both varying degrees of misfolding and decreased raft partitioning which strongly correlate to disease severity. We hypothesize that PMP22 fold stability is necessary for both raft partitioning and trafficking to the plasma membrane. From the screen, we are identifying hits that we sort into different categories based on their effects on (1) PMP22 raft partitioning (2) GPMV phase composition and (3) raft partitioning of other proteins. Identification of small molecules that non-specifically alter raft affinity will be useful tools for dissecting how and why proteins prefer rafts. The identification of small molecules that selectively bind to PMP22 and alter its phase partitioning will be crucial to investigating the relationship between PMP22 structure, raft affinity, and trafficking.

Investigating the organisation of the asymmetric plasma membrane using a novel solvatochromic reporter.

The plasma membrane (PM) of an animal cell is a heterogenous mixture of lipids and proteins that exhibits both lateral heterogeneity and transverse asymmetry in composition. The complex mixture of lipid species with a range of headgroups and degrees of acyl-chain unsaturation influences the properties of the bilayer such as ordering and consequently the function of embedded membrane proteins. In this study, we have probed the presence of liquid-ordered (lo) domains in each leaflet of the PM and their transbilayer registry in live cells by designing a membrane localised probe that is sensitive to lipid order. This solvatochromic probe, C3L, uses Laurdan as a template and reports on changes in lipid order of complex lipid compositions such as Giant Plasma Membrane Vesicles (GPMVs), while exhibiting a preference for lo-domains. C3L distributes across both leaflets in the live cell membrane, regulated by flippases and scramblases and may be selectively and robustly confined to the inner leaflet after back extraction. C3L indicates the inner leaflet to be significantly disordered compared to the outer leaflet wherein the depletion of sphingomyelin (SM) leads to a more outer-leaflet restricted effect but phosphatidylserine (PS) depletion leads to effects on both leaflets, especially the inner leaflet. This suggests that PS and SM play different roles in building and maintaining domains and coupling them across the bilayer. Further, C3L reported lipid organisation indicates domains of smaller scale and broader order distribution in the PM of live cells at the same temperature compared to homogenous and disordered domains in GPMVs derived from them providing support for active de-mixing in membranes of living cells. Using this tool, we intend to explore the impact of membrane composition and cytoskeletal activity in tuning cellular signalling by regulating transbilayer properties of the PM.

Using giant plasma membrane vesicles (GMPVs) to study age-dependent changes in amyloid-β oligomer toxicity.

Aging is a major risk factor associated with several human ailments, including some forms of cancer, autoimmunity, and neurodegeneration. Longitudinal changes in the multicellular organisms, including issues with genomic stability and protein oxidation, have been extensively reported. However, age-dependent changes in plasma membrane lipids and how they contribute to age-related phenotypes are less understood. Moreover, mechanistic understanding about how age-dependent lipidic changes can seed or exacerbate the toxicity of aggregating protein, such as amyloid-β (Aβ) in Alzheimer's disease, is of particular interest. Indeed, certain isolated detergents, fatty acids, and lipids have been shown to nucleate soluble Aβ oligomers (Aβos) with notable cytotoxicity, but little has been done to understand how changes in complex mixtures such as plasma membrane lipids scaffold toxic oligomers in a longitudinal manner. To deconvolute how cellular age can influence the cytotoxic properties of Aβos, giant-plasma membrane derived vesicles (GPMVs) will be collected from cultured cells at various passage numbers and at senescence. Those GMPVs will then be used as lipidic scaffolds for soluble Aβos and the Aβo-GPMV complexes will be treated to cultured cells to monitor their responses. Specifically, Aβo-GPMV toxicity will be monitored by observing changes in cellular respiration, senescence, and/or cell death. The driving hypothesis is that GPMVs derived from aged cells (at high-passage numbers) will seed enhanced toxicity of Aβ oligomers compared to the young (low/medium passage number) counterparts.

FcεRI clusters coupling with signaling condensates and membrane raft domains in mast cell activation.

T cell receptor (TCR) binding to foreign peptides presented by MHC (major histocompatibility complex) triggers T cell activation. Our recent work shows that this T cell activation is promoted by coupling membrane domains with protein condensates containing T cell signal transduction proteins LAT, Grb2, and Sos1. To test whether these observations are generalizable to other cellular systems, we evaluated the coupling of protein condensates to membrane domains in mast cells. Mast cells share many components in common with T cell activation, specifically, LAT, Grb2, and Sos1 are required for mast cell activation downstream of FcεRI clustering induced by multivalent antigens binding IgE. First, using giant plasma membrane vesicles (GPMVs) derived from a model mast cell line (RBL-2H3), we found that FcεRI partitions into the liquid disorder phase prior to antigen stimulation. The addition of a model antigen crosslinks the IgE-FcεRI and changes its partitioning, leading to enrichment in the relatively ordered (lipid raft) phase. Thus, antigen-induced clustering induces FcεRI raft affinity. Concurrently, TIRF imaging showed the formation of LAT+Grb2 condensates after RBL activation, which colocalized with activated FcεRI. These condensates recruited distinct membrane environments, demonstrating the potential coupling of raft domains with FcεRI and protein condensates. Our results indicate that raft coupling with signaling condensates may be a general mechanism for immune cell signaling regulation.

Lipid-mediated interplay between host restriction factor Serinc5 and HIV-1 ENV glycoprotein.

Human immunodeficiency virus (HIV-1) is a pathogenic retrovirus that continues to be a severe threat to public health. HIV-1 is an enveloped virus that enters and infects cells through cell-virus membrane fusion, mediated by HIV-1 envelope glycoprotein, Env.The host restriction factor, Serinc5, impairs this process. It reduces HIV infection rates by incorporating itself into the viral membrane and blocking fusion of the virus with the plasma membrane of uninfected cells. The exact restriction mechanism of Serinc5 remains unknown. Studies suggest that Serinc5 disrupts Env distribution at the membrane and inhibits the protein's fusion function. We hypothesize that Serinc5 acts on Env by altering the physical properties of the surrounding membrane. In this study, we evaluate the nature of the membrane properties affected by Serinc5. Applying the fluorescent probe FLIPPER-TR to measure membrane order in viral particles with and without Serinc5, we discovered that Serinc5 slightly increases lipid order. HIV-1 bearing Env with a truncated cytoplasmic tail (∆CT) avoids infection restriction by Serinc5. We used isolated giant plasma membrane vesicles (GPMVs) to quantify Serinc5 and Env partitioning into specific lipid domains. Our results indicate that Serinc5 and Env partition into the liquid-disordered lipid domains of the plasma membrane. Interestingly, mutant Env ∆CT showed different lipid phase preferences than Serinc5 and wild-type Env. We discuss how these different lipid interactions might affect entry of progeny viruses into cells by membrane fusion.

Lipid scrambling facilitates membrane vesiculation through decreasing membrane stiffness.

Membrane blebbing plays important roles in cell migration, cytokinesis, apoptosis, as well as extracellular vesicle formation for intercellular communication. During budding of the plasma membrane (PM) to form extracellular vesicles, phosphatidylserine is often translocated from the PM inner leaflet to the outer leaflet, indicative of lipid scrambling that results in loss of native membrane asymmetry. Using micrometer-sized giant plasma membrane vesicles (GPMV) as a readout of PM vesiculation, we found that knocking out (KO) the lipid scramblase TMEM16F reduced lipid scrambling and dramatically inhibited GPMV formation, in line with previous observations. TMEM16F repletion could rescue GPMV formation, indicating that lipid scrambling is necessary for membrane budding. We hypothesized three mechanisms to explain this surprising observation, namely that lipid scrambling: (1) depletes PI(4,5)P2 from the inner leaflet leading to detachment of the PM from the actin cytoskeleton, (2) makes PM bilayers softer and easier to bend, (3) externalizes lipids that stabilize highly curved bud necks. Depletion of PI(4,5)P2 was indeed observed during vesiculation, however this occurred in both wild type and KO cells, implying that detachment of PM from actin cytoskeleton due to PIP2 depletion is not sufficient to make large vesicles. Next, through fluorescence lifetime imaging microscopy of a lipid packing reporter (Di4), we found that lipid scrambling leads to a decrease of PM packing, potentially making the membrane softer for bending and thus facilitating membrane budding. Finally, we observed that lipid scrambling resulted in externalization of phosphatidylethanolamine (PE). This PE was preferentially located at the highly curved vesicle necks, suggesting that the intrinsic curvature of PE stabilizes this highly curved membrane structure. Thus, we conclude that lipid scrambling changes membrane biophysical properties, potentially explaining mechanisms underlying vesicle-budding in physiological contexts including apoptotic cell removal, bone mineralization, fertilization, and blood coagulation.

Investigating allosteric regulation by membrane domains of two pentameric ligand-gated ion channels.

Ion channels are embedded in a compositionally heterogeneous membrane with nanoscale structure on the order of ∼10-100 nm. Experiments in giant plasma membrane vesicles suggest that one source of this heterogeneity is that the plasma membrane is poised near to but above a miscibility phase transition. Systems near a phase transition can have high susceptibility, meaning that physical properties are especially sensitive to external perturbations. Previous theoretical work in collaboration with Ben Machta has shown that small changes in the phase transition temperature, Tc, can have a strong influence on ion channel function when channels are coupled by boundary interactions to local changes in lipid composition. Meanwhile, data assembled from literature shows that n-alcohol treatments potentiate GABAA and glycine receptors by an amount that is well predicted by the treatment's corresponding shift in Tc. We hypothesize that these channels are allosterically regulated by local changes in lipid composition. From our model, we expect the channels to have strongly different lipid order preferences in their open versus closed states. In this poster, we present early investigations into this idea using two-color imaging of clustered receptors and membrane domain markers.

Fluorescence Resonance Energy Transfer to Detect Plasma Membrane Perturbations in Giant Plasma Membrane Vesicles.

Disruptions and perturbations of the cellular plasma membrane by peptides have garnered significant interest in the elucidation of biological phenomena. Typically, these complex processes are studied using liposomes as model membranes-either by encapsulating a fluorescent dye or by other spectroscopic approaches, such as nuclear magnetic resonance. Despite incorporating physiologically relevant lipids, no synthetic model truly recapitulates the full complexity and molecular diversity of the plasma membrane. Here, biologically representative membrane models, giant plasma membrane vesicles (GPMVs), are prepared from eukaryotic cells by inducing a budding event with a chemical stressor. The GPMVs are then isolated, and bilayers are labelled with fluorescent lipophilic tracers and incubated in a microplate with a membrane-active peptide. As the membranes become damaged and/or aggregate, the resulting fluorescence resonance energy transfer (FRET) between the two tracers increases and is measured periodically in a microplate. This approach offers a particularly useful way to detect perturbations when the membrane complexity is an important variable to consider. Additionally, it provides a way to kinetically detect damage to the plasma membrane, which can be correlated with the kinetics of peptide self-assembly or structural rearrangements. Key features • Allows testing of various peptide-membrane interaction conditions (peptide:phospholipid ratio, ionic strength, buffer, etc.) at once. • Uses intact plasma membrane vesicles that can be prepared from a variety of cell lines. • Can offer comparable throughput as with traditional synthetic lipid models (e.g., dye-encapsulated liposomes).

Formation of giant plasma membrane vesicles for biological and medical applications: a review

This review involves three main parts: the formation, properties and applications of GPMVs. The applications are also divided into three parts: GPMVs as cell mimics, plasma membrane models and drug carriers.