Plasma Membrane Vesicles Research Articles

Myelin-Associated MAL and PLP Are Unusual among Multipass Transmembrane Proteins in Preferring Ordered Membrane Domains.

Eukaryotic membranes can be partitioned into lipid-driven membrane microdomains called lipid rafts, which function to sort lipids and proteins in the plane of the membrane. As protein selectivity underlies all functions of lipid rafts, there has been significant interest in understanding the structural and molecular determinants of raft affinity. Such determinants have been described for lipids and single-spanning transmembrane proteins; however, how multipass transmembrane proteins (TMPs) partition between ordered and disordered phases has not been widely explored. Here we used cell-derived giant plasma membrane vesicles (GPMVs) to systematically measure multipass TMP partitioning to ordered membrane domains. Across a set of 24 structurally and functionally diverse multipass TMPs, the large majority (92%) had minimal raft affinity. The only exceptions were two myelin-associated four-pass TMPs, myelin and lymphocyte protein (MAL), and proteo lipid protein (PLP). We characterized the potential mechanisms for their exceptional raft affinity and observed that PLP requires cholesterol and sphingolipids for optimal association with ordered membrane domains and that PLP and MAL appear to compete for cholesterol-mediated raft affinity. These observations suggest broad conclusions about the composition of ordered membrane domains in cells and point to previously unrecognized drivers of raft affinity for multipass transmembrane proteins.

Self-Assembly of Mammalian-Cell Membranes on Bioelectronic Devices with Functional Transmembrane Proteins.

Transmembrane proteins (TMPs) regulate processes occurring at the cell surface and are essential gatekeepers of information flow across the membrane. TMPs are difficult to study, given the complex environment of the membrane and its influence on protein conformation, mobility, biomolecule interaction, and activity. For the first time, we create mammalian biomembranes supported on a transparent, electrically conducting polymer surface, which enables dual electrical and optical monitoring of TMP function in its native membrane environment. Mammalian plasma membrane vesicles containing ATP-gated P2X2 ion channels self-assemble on a biocompatible polymer cushion that transduces the changes in ion flux during ATP exposure. This platform maintains the complexity of the native plasma membrane, the fluidity of its constituents, and protein orientation critical to ion channel function. We demonstrate the dual-modality readout using microscopy to characterize protein mobility by single-particle tracking and sensing of ATP gating of P2X2 using electrical impedance spectroscopy. This measurement of TMP activity important for pain sensing, neurological activity, and sensory activity raises new possibilities for drug screening and biosensing applications.

Graphene nanosheets damage the lysosomal and mitochondrial membranes and induce the apoptosis of RBL-2H3 cells

The induced membrane damage is a key mechanism for the cytotoxicity of graphene nanosheets (GNSs). In this research, the physical interaction of GNSs on model membranes was investigated using artificial membranes and plasma membrane vesicles. The effects of the GNSs on plasma membrane, lysosomal and mitochondrial membranes were investigated using rat basophilic leukemia (RBL2H3) cells via lactate dehydrogenase (LDH) assay, acridine orange staining and JC-1 probe, respectively. The physical interaction with model membranes was dominated by electrostatic forces, and the adhered GNSs disrupted the membrane. The degree of physical membrane disruption was quantified by the quartz crystal microbalance with dissipation (QCM-D), confirming the serious membrane disruption. The internalized GNSs were mainly distributed in the lysosomes. They caused plasma membrane leakage, increased the lysosomal membrane permeability (LMP), and depolarized the mitochondrial membrane potential (MMP). The increased cellular levels of reactive oxygen species (ROS) were also detected after GNS exposure. The combination of physical interaction and the excess ROS production damaged the plasma and organelle membranes in living RBL-2H3 cells. The lysosomal and mitochondrial dysfunction, and the oxidative stress further induced cell apoptosis. Specially, the exposure to 25 mg/L GNSs caused severest cell mortality, plasma membrane damage, ROS generation, MMP depolarization and apoptosis. The research findings provide more comprehensive information on the graphene-induced plasma and organelle membrane damage, which is important to understand and predict the cytotoxicity of carbon-based nanomaterials.

Nanodomains can persist at physiologic temperature in plasma membrane vesicles and be modulated by altering cell lipids

The formation and properties of liquid-ordered (Lo) lipid domains (rafts) in the plasma membrane are still poorly understood. This limits our ability to manipulate ordered lipid domain-dependent biological functions. Giant plasma membrane vesicles (GPMVs) undergo large-scale phase separations into coexisting Lo and liquid-disordered lipid domains. However, large-scale phase separation in GPMVs detected by light microscopy is observed only at low temperatures. Comparing Förster resonance energy transfer-detected versus light microscopy-detected domain formation, we found that nanodomains, domains of nanometer size, persist at temperatures up to 20°C higher than large-scale phases, up to physiologic temperature. The persistence of nanodomains at higher temperatures is consistent with previously reported theoretical calculations. To investigate the sensitivity of nanodomains to lipid composition, GPMVs were prepared from mammalian cells in which sterol, phospholipid, or sphingolipid composition in the plasma membrane outer leaflet had been altered by cyclodextrin-catalyzed lipid exchange. Lipid substitutions that stabilize or destabilize ordered domain formation in artificial lipid vesicles had a similar effect on the thermal stability of nanodomains and large-scale phase separation in GPMVs, with nanodomains persisting at higher temperatures than large-scale phases for a wide range of lipid compositions. This indicates that it is likely that plasma membrane nanodomains can form under physiologic conditions more readily than large-scale phase separation. We also conclude that membrane lipid substitutions carried out in intact cells are able to modulate the propensity of plasma membranes to form ordered domains. This implies lipid substitutions can be used to alter biological processes dependent upon ordered domains.

Patches and Blebs: A Comparative Study of the Composition and Biophysical Properties of Two Plasma Membrane Preparations from CHO Cells.

This study was aimed at preparing and characterizing plasma membranes (PM) from Chinese Hamster Ovary (CHO) cells. Two methods of PM preparation were applied, one based on adhering cells to a poly-lysine-coated surface, followed by hypotonic lysis and removal of intracellular components, so that PM patches remain adhered to each other, and a second one consisting of bleb induction in cells, followed by separation of giant plasma membrane vesicles (GPMV). Both methods gave rise to PM in sufficient amounts to allow biophysical and biochemical characterization. Laurdan generalized polarization was used to measure molecular order in membranes, PM preparations were clearly more ordered than the average cell membranes (GP ≈0.450 vs. ≈0.20 respectively). Atomic force microscopy was used in the force spectroscopy mode to measure breakthrough forces of PM, both PM preparations provided values in the 4–6 nN range, while the corresponding value for whole cell lipid extracts was ≈2 nN. Lipidomic analysis of the PM preparations revealed that, as compared to the average cell membranes, PM were enriched in phospholipids containing 30–32 C atoms in their acyl chains but were relatively poor in those containing 34–40 C atoms. PM contained more saturated and less polyunsaturated fatty acids than the average cell membranes. Blebs (GPMV) and patches were very similar in their lipid composition, except that blebs contained four-fold the amount of cholesterol of patches (≈23 vs. ≈6 mol% total membrane lipids) while the average cell lipids contained 3 mol%. The differences in lipid composition are in agreement with the observed variations in physical properties between PM and whole cell membranes.

Uncovering The Unique Functions And Regulation Of The Palmitoylated Calcineurin Isoform, CNβ1

Calcineurin (CN), the serine/threonine protein phosphatase and the target of immunosuppressants FK506 and CysA, is a key regulator of Ca2+ signaling with critical roles in the immune, cardiovascular and nervous systems. CN is a heterodimer of regulatory and catalytic subunits whose functions and regulation by Ca2+ and calmodulin are well understood for canonical CN isozymes (CNalpha, CNβ2). In contrast, the CNβ1 isozyme, contains a catalytic subunit with a non‐canonical C‐terminus generated by alternative 3’ pre‐mRNA processing. The few studies of CNβ1, conserved in eukaryotes and broadly expressed in human tissues, demonstrate its unique physiological functions. For example, overexpression of the CNβ2 promotes cardiac hypertrophy through activation of NFAT, whereas CNβ1 is cardioprotective and does not dephosphorylate NFAT. However, CNβ1 specific substrates remain unknown.We determined that the unique C‐tail confers distinct regulatory properties, intracellular localization and function to CNβ1. In vitro, CNβ1 displays distinct enzymatic properties. Instead of the auto‐inhibitory domain that blocks the active site of canonical CN isoforms under non‐signaling conditions, CNβ1 is autoinhibited by a sequence motif at its C` tail that blocks substrate binding. We show that CNβ1 localizes to the plasma membrane (PM), Golgi, and intracellular vesicles, in contrast to the cytosolic CNβ2, due to the palmitoylation of two conserved cysteine residues unique to its C‐tail. Palmitoylation allows CNβ1 to access substrates that are distinct from canonical CN isoforms. CNβ1 preferentially interacts with membrane proteins, and all members of a highly conserved PI4‐kinase complex, PI4KIIIA, TTC7B, FAM126A and EFR3B, were identified as CNβ1‐specific interactors by AP‐MS. This complex recruits the cytosolic PI4KIIIA to the PM where it synthesizes phosphatidylinositol‐4‐phosphate (PI4P), a precursor of the critical signaling phospholipid, PI(4,5)P2, required for sustained Ca2+ signaling through GPCRs. We identify a CN binding motif in FAM126A, mutation of which results in FAM126A hyperphosphorylation, and prevents association of CNβ1 with the PI4KIIIA complex. Using BRET‐based detection of phosphoinositides in live cells, we show that CN promotes PI4P synthesis at the PM during signaling through the muscarinic receptor, and hypothesize that CNβ1 mediates this regulation by dephosphorylating FAM126A at the PM.Using pulse‐chase analysis in cells metabolically labelled with a palmitate analog, we demonstrate that palmitoylation of CNβ1 is dynamic and turns over rapidly. We are currently investigating whether dynamic palmitoylation regulates localization and activity CNβ1 activity in vivo during signaling. Together, these studies provide the first mechanistic understanding of CNβ1 and not only uncovers a novel Ca2+‐dependent mechanism for activation of PI4P synthesis at the PM during signaling but also suggests palmitoylation as a novel mechanism that confers spatio/temporal regulation of calcineurin signaling in cells.Support or Funding InformationNIGMS

Protein Kinase A (PKA) Catalytic Subunits a and b Have Non‐Redundant Functions in Collecting Duct Cells

Vasopressin regulates osmotic water transport in the renal collecting duct by PKA‐mediated control of the water channel aquaporin‐2 (AQP2). Collecting duct principal cells express two seemingly redundant PKA catalytic subunits, PKA catalytic α (PKA‐Cα [Gene symbol: Prkaca]) and PKA catalytic β (PKA‐Cβ [Gene symbol: Prkacb]). To identify the relative roles of these two proteins, we carried out deep phosphoproteomic analysis in cultured mpkCCD cells in which either PKA‐Cα (n=3) or PKA‐Cβ (n=3) was deleted using CRISPR‐Cas9‐based genome editing (generated in PMID 28973931). Cells were grown on permeable supports in the presence of the vasopressin analog dDAVP (0.1nM). Controls were cells carried through the genome editing procedure, but without deletion of PKA. Quantification used TMT mass tags in an Orbitrap Fusion Lumos ETD mass spectrometer. Preliminary measures of total protein amounts showed that PKA‐Cα deletion resulted in a near disappearance of AQP2 protein, while PKA‐Cβ deletion was associated with an increase in AQP2. A total of 4635 phosphopeptides were quantified (Figure 1). The red data points represent phosphopeptides that correspond to previously identified PKA target sites. Stringent statistical criteria (Pmod < 0.01 and |log2(KO/control)| > 0.503) revealed that 67 phosphopeptides were significantly altered with PKA‐Cα deletion, while 21 phosphopeptides were significantly altered with PKA‐Cβ deletion. Only four sites were changed in both. We used Gene Ontology (GO‐CC) Cellular Component terms to identify sets of target phosphoproteins localized in different regions of the cell. The target proteins identified in PKA‐Cα‐null cells were largely associated with cell membranes and membrane vesicles, while target proteins in the PKA‐Cβ‐null cells were largely associated with the actin cytoskeleton and cell junctions. In summary, PKA‐Cα and PKA‐Cβ appear to serve substantially different functions in renal collecting duct cells. The differences in the phosphorylation targets of PKA‐Cα and PKA‐Cβ suggest differences in the cellular localization of the two PKA subunits. These findings in addition to the dramatically different effect of PKA‐Cα and PKA‐Cβ deletion on AQP2 protein abundance support the conclusion that PKA‐Cα and PKA‐Cβ have non‐redundant functions in collecting duct cells.Support or Funding InformationK.S. was supported by the Biomedical Engineering Summer Internship Program at NIH and carried out all of the mass spectrometry experiments. Work was supported by NHLBI Project ZIA‐HL001285.Volcano plots showing effect of PKA‐Cα deletion (left) and PKA‐Cβ deletion (right) on phosphoproteome of mouse mpkCCD cells. Three biological replicates for each.Figure 1

From algal cells to autofluorescent ghost plasma membrane vesicles

Plasma membrane vesicles can be effective, non-toxic carriers for microscale material transport, provide a convenient model for probing membrane-related processes, since intracellular biochemical processes are eliminated. We describe here a fine-tuned protocol for isolating ghost plasma membrane vesicles from the unicellular alga Dunaliella tertiolecta, and preliminary characterization of their structural features and permeability properties, with comparisons to giant unilamellar phospholipid vesicles. The complexity of the algal ghost membrane vesicles reconstructed from the native membrane material released after hypoosmotic stress lies between that of phospholipid vesicles and cells. AFM structural characterization of reconstructed vesicles shows a thick envelope and a nearly empty vesicle interior. The surface of the envelope contains a heterogeneous distribution of densely packed, nanometer-scale globules and pore-like structures which may be derived from surface coat proteins. Confocal fluorescence imaging reveals the highly pigmented photosynthetic apparatus located within the thylakoid membrane and retained in the vesicle membrane. Transport of the fluorescent dye calcein into ghost and giant unilamellar vesicles reveals significant differences in permeability. Expanded knowledge of this unique membrane system will contribute to the design of marine bio-inspired carriers for advanced biotechnological applications.

Membrane Protein Dimerization in Cell-Derived Lipid Membranes Measured by FRET with MC Simulations

Many membrane proteins are thought to function as dimers or higher oligomers, but measuring membrane protein oligomerization in lipid membranes is particularly challenging. Förster resonance energy transfer (FRET) and fluorescence cross-correlation spectroscopy are noninvasive, optical methods of choice that have been applied to the analysis of dimerization of single-spanning membrane proteins. However, the effects inherent to such two-dimensional systems, such as the excluded volume of polytopic transmembrane proteins, proximity FRET, and rotational diffusion of fluorophore dipoles, complicate interpretation of FRET data and have not been typically accounted for. Here, using FRET and fluorescence cross-correlation spectroscopy, we introduce a method to measure surface protein density and to estimate the apparent Förster radius, and we use Monte Carlo simulations of the FRET data to account for the proximity FRET effect occurring in confined two-dimensional environments. We then use FRET to analyze the dimerization of human rhomboid protease RHBDL2 in giant plasma membrane vesicles. We find no evidence for stable oligomers of RHBDL2 in giant plasma membrane vesicles of human cells even at concentrations that highly exceed endogenous expression levels. This indicates that the rhomboid transmembrane core is intrinsically monomeric. Our findings will find use in the application of FRET and fluorescence correlation spectroscopy for the analysis of oligomerization of transmembrane proteins in cell-derived lipid membranes.

Role of Proton Motive Force in Photoinduction of Cytoplasmic Streaming in Vallisneria Mesophyll Cells.

In mesophyll cells of the aquatic monocot Vallisneria, red light induces rotational cytoplasmic streaming, which is regulated by the cytoplasmic concentration of Ca2+. Our previous investigations revealed that red light induces Ca2+ efflux across the plasma membrane (PM), and that both the red light-induced cytoplasmic streaming and the Ca2+ efflux are sensitive to vanadate, an inhibitor of P-type ATPases. In this study, pharmacological experiments suggested the involvement of PM H+-ATPase, one of the P-type ATPases, in the photoinduction of cytoplasmic streaming. We hypothesized that red light would activate PM H+-ATPase to generate a large H+ motive force (PMF) in a photosynthesis-dependent manner. We demonstrated that indeed, photosynthesis increased the PMF and induced phosphorylation of the penultimate residue, threonine, of PM H+-ATPase, which is a major activation mechanism of H+-ATPase. The results suggested that a large PMF generated by PM H+-ATPase energizes the Ca2+ efflux across the PM. As expected, we detected a putative Ca2+/H+ exchange activity in PM vesicles isolated from Vallisneria leaves.

Populus euphratica remorin 6.5 activates plasma membrane H+-ATPases to mediate salt tolerance.

Remorins (REMs) play an important role in the ability of plants to adapt to adverse environments. PeREM6.5, a protein of the REM family in Populus euphratica (salt-resistant poplar), was induced by NaCl stress in callus, roots and leaves. We cloned the full-length PeREM6.5 from P. euphratica and transformed it into Escherichia coli and Arabidopsis thaliana. PeREM6.5 recombinant protein significantly increased the H+-ATPase hydrolytic activity and H+ transport activity in P. euphratica plasma membrane (PM) vesicles. Yeast two-hybrid assay showed that P. euphratica REM6.5 interacted with RPM1-interacting protein 4 (PeRIN4). Notably, the PeREM6.5-induced increase in PM H+-ATPase activity was enhanced by PeRIN4 recombinant protein. Overexpression of PeREM6.5 in Arabidopsis significantly improved salt tolerance in transgenic plants in terms of survival rate, root growth, electrolyte leakage and malondialdehyde content. Arabidopsis plants overexpressing PeREM6.5 retained high PM H+-ATPase activity in both in vivo and in vitro assays. PeREM6.5-transgenic plants had reduced accumulation of Na+ due to the Na+ extrusion promoted by the H+-ATPases. Moreover, the H+ pumps caused hyperpolarization of the PM, which reduced the K+ loss mediated by the depolarization-activated channels in the PM of salinized roots. Therefore, we conclude that PeREM6.5 regulated H+-ATPase activity in the PM, thus enhancing the plant capacity to maintain ionic homeostasis under salinity.

Paclitaxel-Loaded Macrophage Membrane Camouflaged Albumin Nanoparticles for Targeted Cancer Therapy.

BackgroundMelanoma is the most common symptom of aggressive skin cancer, and it has become a serious health concern worldwide in recent years. The metastasis rate of malignant melanoma remains high, and it is highly difficult to cure with the currently available treatment options. Effective yet safe therapeutic options are still lacking. Alternative treatment options are in great demand to improve the therapeutic outcome against advanced melanoma. This study aimed to develop albumin nanoparticles (ANPs) coated with macrophage plasma membranes (RANPs) loaded with paclitaxel (PTX) to achieve targeted therapy against malignant melanoma.MethodsMembrane derivations were achieved by using a combination of hypotonic lysis, mechanical membrane fragmentation, and differential centrifugation to empty the harvested cells of their intracellular contents. The collected membrane was then physically extruded through a 400 nm porous polycarbonate membrane to form macrophage cell membrane vesicles. Albumin nanoparticles were prepared through a well-studied nanoprecipitation process. At last, the two components were then coextruded through a 200 nm porous polycarbonate membrane.ResultsUsing paclitaxel as the model drug, PTX-loaded RANPs displayed significantly enhanced cytotoxicity and apoptosis rates compared to albumin nanoparticles without membrane coating in the murine melanoma cell line B16F10. RANPs also exhibited significantly higher internalization efficiency in B16F10 cells than albumin nanoparticles without a membrane coating. Next, a B16F10 tumor xenograft mouse model was established to explore the biodistribution profiles of RANPs, which showed prolonged blood circulation and selective accumulation at the tumor site. PTX-loaded RANPs also demonstrated greatly improved antitumor efficacy in B16F10 tumor-bearing mouse xenografts.ConclusionAlbumin-based nanoscale delivery systems coated with macrophage plasma membranes offer a highly promising approach to achieve tumor-targeted therapy following systemic administration.

Cell Membrane-Coated Nanoparticles: Research Advances

Cell membranes have been continuously imitated and used for the modification of nanoparticles (NPs) to improve NP biological properties. Cell membrane-coated NPs, where core NPs are wrapped with plasma membrane vesicles, show high biocompatibility, targeting specificity and low side effects. Compared with conventional strategies, this novel approach directly leverages intact and natural functions of cell membranes, instead of replicating these features via synthetic techniques. This top-down technique bestows NPs with enhanced biointerfacing capabilities with potential in the diagnosis and treatment of cancer, infection and other diseases. Herein, we report on the advances in cell membrane-coated NPs, including the preparation process, source cell membranes for wrapping and potential applications of these cell membrane-coated NPs.

Giant Endoplasmic Reticulum vesicles (GERVs), a novel model membrane tool

Artificial giant vesicles have proven highly useful as membrane models in a large variety of biophysical and biochemical studies. They feature accessibility for manipulation and detection, but lack the compositional complexity needed to reconstitute complicated cellular processes. For the plasma membrane (PM), this gap was bridged by the establishment of giant PM vesicles (GPMVs). These native membranes have facilitated studies of protein and lipid diffusion, protein interactions, electrophysiology, fluorescence analysis of lateral domain formation and protein and lipid partitioning as well as mechanical membrane properties and remodeling. The endoplasmic reticulum (ER) is key to a plethora of biological processes in any eukaryotic cell. However, its intracellular location and dynamic and intricate tubular morphology makes it experimentally even less accessible than the PM. A model membrane, which will allow the afore-mentioned types of studies on GPMVs to be performed on ER membranes outside the cell, is therefore genuinely needed. Here, we introduce the formation of giant ER vesicles, termed GERVs, as a new tool for biochemistry and biophysics. To obtain GERVs, we have isolated ER membranes from Saccharomyces cerevisiae and fused them by exploiting the atlastin-like fusion protein Sey1p. We demonstrate the production of GERVs and their utility for further studies.

Creating Supported Plasma Membrane Bilayers Using Acoustic Pressure.

Model membrane systems are essential tools for the study of biological processes in a simplified setting to reveal the underlying physicochemical principles. As cell-derived membrane systems, giant plasma membrane vesicles (GPMVs) constitute an intermediate model between live cells and fully artificial structures. Certain applications, however, require planar membrane surfaces. Here, we report a new approach for creating supported plasma membrane bilayers (SPMBs) by bursting cell-derived GPMVs using ultrasound within a microfluidic device. We show that the mobility of outer leaflet molecules is preserved in SPMBs, suggesting that they are accessible on the surface of the bilayers. Such model membrane systems are potentially useful in many applications requiring detailed characterization of plasma membrane dynamics.

Keeping in touch with the membrane; protein- and lipid-mediated confinement of caveolae to the cell surface.

Caveolae are small Ω-shaped invaginations of the plasma membrane that play important roles in mechanosensing, lipid homeostasis and signaling. Their typical morphology is characterized by a membrane funnel connecting a spherical bulb to the membrane. Membrane funnels (commonly known as necks and pores) are frequently observed as transient states during fusion and fission of membrane vesicles in cells. However, caveolae display atypical dynamics where the membrane funnel can be stabilized over an extended period of time, resulting in cell surface constrained caveolae. In addition, caveolae are also known to undergo flattening as well as short-range cycles of fission and fusion with the membrane, requiring that the membrane funnel closes or opens up, respectively. This mini-review considers the transition between these different states and highlights the role of the protein and lipid components that have been identified to control the balance between surface association and release of caveolae.

Plant plasma membrane vesicles interaction with keratinocytes reveals their potential as carriers

During the last few years, membrane vesicles (as exovesicles) have emerged as potential nanocarriers for therapeutic applications. They are receiving attention due to their proteo-lipid nature, size, biocompatibility and biodegradability. In this work, we investigated the potential use of isolated root plasma membrane vesicles from broccoli plants as nanocarriers. For that, the entrapment efficiency and integrity of the vesicles were determined. Also, the delivery of keratinocytes and penetrability through skin were studied. The results show that the broccoli vesicles had high stability, in relation to their proteins, and high entrapment efficiency. Also, the interaction between the vesicles and keratinocytes was proven by the delivery of an encapsulated fluorescent product into cells and by the detection of plant proteins in the keratinocyte plasma membrane, showing the interactions between the membranes of two species of distinct biological kingdoms. Therefore, these results, together with the capacity of brassica vesicles to cross the skin layers, detected by fluorescent penetration, enable us to propose a type of nanocarrier obtained from natural plant membranes for use in transdermal delivery.

Impact of Nanoscale Hindrances on the Relationship between Lipid Packing and Diffusion in Model Membranes.

Membrane models have allowed for precise study of the plasma membrane’s biophysical properties, helping to unravel both structural and dynamic motifs within cell biology. Freestanding and supported bilayer systems are popular models to reconstitute membrane-related processes. Although it is well-known that each have their advantages and limitations, comprehensive comparison of their biophysical properties is still lacking. Here, we compare the diffusion and lipid packing in giant unilamellar vesicles, planar and spherical supported membranes, and cell-derived giant plasma membrane vesicles. We apply florescence correlation spectroscopy (FCS), spectral imaging, and super-resolution stimulated emission depletion FCS to study the diffusivity, lipid packing, and nanoscale architecture of these membrane systems, respectively. Our data show that lipid packing and diffusivity is tightly correlated in freestanding bilayers. However, nanoscale interactions in the supported bilayers cause deviation from this correlation. These data are essential to develop accurate theoretical models of the plasma membrane and will serve as a guideline for suitable model selection in future studies to reconstitute biological processes.

Nanodomains Persist to much Higher Temperatures than Large Scale Phase Separation in Giant Plasma Membrane Vesicles and Can Respond Differently to Alterations of Plasma Membrane Lipid Composition

The formation and properties of liquid ordered (Lo) lipid domains (rafts) in the plasma membrane is still poorly understood. Giant plasma membrane vesicles (GPMV) derived from mammalian cells can undergo large scale phase separations into co-existing Lo and disordered lipid (Ld) domains with properties similar to domains in artificial lipid vesicles containing simple lipid mixtures. However, large scale phase separation in GPMV detected by light microscopy only occurs at low temperature. We compared large domain formation detected by light microscopy to nanodomain formation in GPMV using a FRET assay. We found that nanodomains can persist to physiological temperatures, much higher temperatures than large scale phase separation. Altering sterol or phospholipid structure in the plasma membrane outer leaflet of intact mammalian cells by cyclodextrin-catalyzed lipid exchange altered both GPMV lipid composition and the ability of GPMV to form ordered domains. Phospholipids and sterols that either stabilize or destabilize ordered domain formation in artificial lipid vesicles had a similar effect on ordered domain stability in GPMV. Nanodomain and large scale domain formation were generally stabilized or destabilized by lipid substitutions to a similar (but not identical) extent. These results show it is possible to alter the lipid composition and properties of membrane vesicles derived from plasma membrane. Other experiments found that after transfection and expression of soluble GFP in mammalian cells, GPMVs contained GFP in their lumen. Because membrane vesicles can be used to deliver biomolecules to cells, combining an analogous expression step with lipid exchange might allow preparation of plasma membrane vesicles with improved abilities to deliver cytosolic molecules to other cells.

Cell-Derived Plasma Membrane Vesicles are Permeable to Hydrophilic Macromolecules

Giant Plasma Membrane Vesicles (GPMVs) are a widely used model system for biochemical and biophysical analysis of the isolated mammalian plasma membrane (PM). A core advantage of these vesicles is that they maintain the native lipid and protein diversity of the plasma membrane while affording the experimental flexibility of synthetic giant vesicles. In addition to fundamental investigations of PM structure and composition, GPMVs have been used to evaluate the binding of proteins and small molecules to cell-derived membranes, and the permeation of drug-like molecules through them.