Simultaneous membrane and RNA binding by tick-borne encephalitis virus capsid protein.

Tick-borne encephalitis virus is an enveloped, pathogenic, RNA virus in the family Flaviviridae, genus Flavivirus. Viral particles are formed when the nucleocapsid, consisting of an RNA genome and multiple copies of the capsid protein, buds through the endoplasmic reticulum membrane and acquires the viral envelope and the associated proteins. The coordination of the nucleocapsid components to the sites of assembly and budding are poorly understood. Here, we investigate the interactions of the wild-type and truncated capsid proteins with membranes with biophysical methods and model membrane systems. We show that capsid protein initially binds membranes via electrostatic interactions with negatively-charged lipids, which is followed by membrane insertion. Additionally, we show that membrane-bound capsid protein can recruit viral genomic RNA. We confirm the biological relevance of the biophysical findings by using mass spectrometry to show that purified virions contain negatively-charged lipids. Our results suggest that nucleocapsid assembly is coordinated by negatively-charged membrane patches on the endoplasmic reticulum and that the capsid protein mediates direct contacts between the nucleocapsid and the membrane.

Probing the Separation Distance between Biological Nanoparticles and Cell Membrane Mimics Using Neutron Reflectometry with Sub-Nanometer Accuracy.

Nanoparticle interactions with cellular membranes are controlled by molecular recognition reactions and regulate a multitude of biological processes, including virus infections, biological nanoparticle-mediated cellular communication, and drug delivery applications. Aided by the design of various supported cell membrane mimics, multiple methods have been employed to investigate these types of interactions, revealing information on nanoparticle coverage, interaction kinetics, as well as binding strength; however, precise quantification of the separation distance across which these delicate interactions occur remains elusive. Here, we demonstrate that carefully designed neutron reflectometry (NR) experiments followed by an attentive selection and application of suitable theoretical models offer a means to quantify the distance separating biological nanoparticles from a supported lipid bilayer (SLB) with sub-nanometer precision. The distance between the nanoparticles and SLBs was tuned by exploiting either direct adsorption or specific binding using DNA tethers with different conformations, revealing separation distances of around 1, 3, and 7 nm with nanometric accuracy. We also show that NR provides precise information on nanoparticle coverage, size distribution, material composition, and potential structural changes in the underlying planar SLB induced upon nanoparticle binding. The precision with which these parameters could be quantified should pave an attractive path for investigations of the interactions between nanoparticles and interfaces at length scales and resolutions that were previously inaccessible. This thus makes it possible to, for example, gain an in-depth understanding of the molecular recognition reactions of inorganic and biological nanoparticles with cellular membranes.

Membrane insertion mechanism of the caveola coat protein Cavin1.

Caveolae are small plasma membrane invaginations, important for control of membrane tension, signaling cascades, and lipid sorting. The caveola coat protein Cavin1 is essential for shaping such high curvature membrane structures. Yet, a mechanistic understanding of how Cavin1 assembles at the membrane interface is lacking. Here, we used model membranes combined with biophysical dissection and computational modeling to show that Cavin1 inserts into membranes. We establish that initial phosphatidylinositol (4, 5) bisphosphate [PI(4,5)P2]-dependent membrane adsorption of the trimeric helical region 1 (HR1) of Cavin1 mediates the subsequent partial separation and membrane insertion of the individual helices. Insertion kinetics of HR1 is further enhanced by the presence of flanking negatively charged disordered regions, which was found important for the coassembly of Cavin1 with Caveolin1 in living cells. We propose that this intricate mechanism potentiates membrane curvature generation and facilitates dynamic rounds of assembly and disassembly of Cavin1 at the membrane.

Protein-lipid interaction at low pH induces oligomerization of the MakA cytotoxin from Vibrio cholerae.

The α-pore-forming toxins (α-PFTs) from pathogenic bacteria damage host cell membranes by pore formation. We demonstrate a remarkable, hitherto unknown mechanism by an α-PFT protein from Vibrio cholerae. As part of the MakA/B/E tripartite toxin, MakA is involved in membrane pore formation similar to other α-PFTs. In contrast, MakA in isolation induces tube-like structures in acidic endosomal compartments of epithelial cells in vitro. The present study unravels the dynamics of tubular growth, which occurs in a pH-, lipid-, and concentration-dependent manner. Within acidified organelle lumens or when incubated with cells in acidic media, MakA forms oligomers and remodels membranes into high-curvature tubes leading to loss of membrane integrity. A 3.7 Å cryo-electron microscopy structure of MakA filaments reveals a unique protein-lipid superstructure. MakA forms a pinecone-like spiral with a central cavity and a thin annular lipid bilayer embedded between the MakA transmembrane helices in its active α-PFT conformation. Our study provides insights into a novel tubulation mechanism of an α-PFT protein and a new mode of action by a secreted bacterial toxin.

A tripartite cytolytic toxin formed by Vibrio cholerae proteins with flagellum-facilitated secretion.

The protein MakA was discovered as a motility-associated secreted toxin from Vibrio cholerae Here, we show that MakA is part of a gene cluster encoding four additional proteins: MakB, MakC, MakD, and MakE. MakA, MakB, and MakE were readily detected in culture supernatants of wild-type V. cholerae, whereas secretion was very much reduced from a flagellum-deficient mutant. Crystal structures of MakA, MakB, and MakE revealed a structural relationship to a superfamily of bacterial pore-forming toxins. Expression of MakA/B/E in Escherichia coli resulted in toxicity toward Caenorhabditis elegans used as a predatory model organism. None of these Mak proteins alone or in pairwise combinations were cytolytic, but an equimolar mixture of MakA, MakB, and MakE acted as a tripartite cytolytic toxin in vitro, causing lysis of erythrocytes and cytotoxicity on cultured human colon carcinoma cells. Formation of oligomeric complexes on liposomes was observed by electron microscopy. Oligomer interaction with membranes was initiated by MakA membrane binding followed by MakB and MakE joining the assembly of a pore structure. A predicted membrane insertion domain of MakA was shown by site-directed mutagenesis to be essential for toxicity toward C. elegans Bioinformatic analyses revealed that the makCDBAE gene cluster is present as a genomic island in the vast majority of sequenced genomes of V. cholerae and the fish pathogen Vibrio anguillarum We suggest that the hitherto-unrecognized cytolytic MakA/B/E toxin can contribute to Vibrionaceae fitness and virulence potential in different host environments and organisms.

Formation of Supported Lipid Bilayers Derived from Vesicles of Various Compositional Complexity on Conducting Polymer/Silica Substrates.

Supported lipid bilayers (SLBs) serve important roles as minimalistic models of cellular membranes in multiple diagnostic and pharmaceutical applications as well as in the strive to gain fundamental insights about their complex biological function. To further expand the utility of SLBs, there is a need to go beyond simple lipid compositions to thereby better mimic the complexity of native cell membranes, while simultaneously retaining their compatibility with a versatile range of analytical platforms. To meet this demand, we have in this work explored SLB formation on PEDOT:PSS/silica nanoparticle composite films and mesoporous silica films, both capable of transporting ions to an underlying conducting PEDOT:PSS film. The SLB formation process was evaluated by using the quartz crystal microbalance with dissipation (QCM-D) monitoring, total internal reflection fluorescence (TIRF) microscopy, and fluorescence recovery after photobleaching (FRAP) for membranes made of pure synthetic lipids with or without the reconstituted membrane protein ß-secretase 1 (BACE1) as well as cell-derived native lipid vesicles containing overexpressed BACE1. The mesoporous silica thin film was superior to the PEDOT:PSS/silica nanoparticle composite, providing successful formation of bilayers with high lateral mobility and low defect density even for the most complex native cell membranes.

FRET-Based Assay for the Quantification of Extracellular Vesicles and Other Vesicles of Complex Composition.

Research in the field of extracellular vesicles is rapidly expanding and finding footholds in many areas of medical science. However, the availability of methodologies to quantify the concentration of membrane material present in a sample remains limited. Herein, we present a novel approach for the quantification of vesicle material, specifically the quantification of the total lipid membrane surface area, found in a sample using Förster resonance energy transfer (FRET). In this assay, sonication is used to drive the fusion between vesicles in the sample to be quantified and liposomes containing a pair of FRET fluorophores. The change in emission spectrum upon vesicle fusion is directly related to the total membrane surface area of the sample added, and a calibration curve allows for the quantification of a variety of vesicle species, including enveloped viruses, bacterial outer membrane vesicles, and mammalian extracellular vesicles. Without extensive optimization of experimental parameters, we were able to quantify down to ∼109 vesicles/mL, using as little as 60 µL of the sample. The assay precision was comparable to that of a commercial nanoparticle tracking analysis system. While its limit of detection was slightly higher, the FRET assay is superior for the detection of small vesicles, as its performance is vesicle-size-independent. Taken together, the FRET assay is a simple, robust, and versatile method for the quantification of a variety of purified vesicle samples.

Lipid vesicle composition influences the incorporation and fluorescence properties of the lipophilic sulphonated carbocyanine dye SP-DiO.

Lipophilic carbocyanine dyes are widely used as fluorescent cell membrane probes in studies ranging from biophysics to cell biology. While they are extremely useful for qualitative observation of lipid structures, a major problem impairing quantitative studies is that the chemical environment of the lipid bilayer affects both the dye's insertion efficiency and photophysical properties. We present a systematic investigation of the sulphonated carbocyanine dye 3,3'-dioctadecyl-5,5'-di(4-sulfophenyl) (SP-DiO) and demonstrate how its insertion efficiency into pre-formed lipid bilayers and its photophysical properties therein determine its apparent fluorescence intensity in different lipid environments. For this purpose, we use large unilamellar vesicles (LUVs) made of lipids with distinct chain unsaturation, acyl chain length, head group charge, and with variation in membrane cholesterol content as models. Using a combination of absorbance, fluorescence emission, and fluorescence lifetime measurements we reveal that SP-DiO incorporates more efficiently into liquid disordered phases compared to gel phases. Moreover, incorporation into the latter phase is most efficient when the mismatch between the length of the lipid and dye hydrocarbon chains is small. Furthermore, SP-DiO incorporation is less efficient in LUVs composed of negatively charged lipids. Lastly, when cholesterol was included in the LUV membranes, we observed significant spectral shifts, consistent with dye aggregation. Taken together, our study highlights the complex interplay between membrane composition and labeling efficiency with lipophilic dyes and advocates for careful assessment of fluorescence data when attempting a quantitative analysis of fluorescence data with such molecules.

Structure and Composition of Native Membrane Derived Polymer-Supported Lipid Bilayers.

Over the last two decades, supported lipid bilayers (SLBs) have been extensively used as model systems to study cell membrane structure and function. While SLBs have been traditionally produced from simple lipid mixtures, there has been a recent surge in compositional complexity to better mimic cellular membranes and thereby bridge the gap between classic biophysical approaches and cell experiments. To this end, native cellular membrane derived SLBs (nSLBs) have emerged as a new category of SLBs. As a new type of biomimetic material, an analytical workflow must be designed to characterize its molecular composition and structure. Herein, we demonstrate how a combination of fluorescence microscopy, neutron reflectometry, and secondary ion mass spectrometry offers new insights on structure, composition, and quality of nSLB systems formed using so-called hybrid vesicles, which are a mixture of native membrane material and synthetic lipids. With this approach, we demonstrate that the nSLB formed a continuous structure with complete mixing of the synthetic and native membrane components and a molecular stoichiometry that essentially mirrors that of the hybrid vesicles. Furthermore, structural investigation of the nSLB revealed that PEGylated lipids do not significantly thicken the hydration layer between the bilayer and substrate when on silicon substrates; however, nSLBs do have more topology than their simpler, purely synthetic counterparts. Beyond new insights regarding the structure and composition of nSLB systems, this work also serves to guide future researchers in producing and characterizing nSLBs from their cellular membrane of choice.

Spatiotemporal Kinetics of Supported Lipid Bilayer Formation on Glass via Vesicle Adsorption and Rupture.

Supported lipid bilayers (SLBs) represent one of the most popular mimics of the cell membrane. Herein, we have used total internal reflection fluorescence microscopy for in-depth characterization of the vesicle-mediated SLB formation mechanism on a common silica-rich substrate, borosilicate glass. Fluorescently labeling a subset of vesicles allowed us to monitor the adsorption of individual labeled vesicles, resolve the onset of SLB formation from small seeds of SLB patches, and track their growth via SLB-edge-induced autocatalytic rupture of adsorbed vesicles. This made it possible to perform the first quantitative measurement of the SLB front velocity, which is shown to increase up to 1 order of magnitude with time. This effect can be classified as dramatic because in many other physical, chemical, or biological kinetic processes the front velocity is either constant or decreasing with time. The observation was successfully described with a theoretical model and Monte Carlo simulations implying rapid local diffusion of lipids upon vesicle rupture.

Cell Membrane Derived Platform To Study Virus Binding Kinetics and Diffusion with Single Particle Sensitivity.

Discovery and development of new antiviral therapies essentially rely on two key factors: an in-depth understanding of the mechanisms involved in viral infection and the development of fast and versatile drug screening platforms. To meet those demands, we present a biosensing platform to probe virus-cell membrane interactions on a single particle level. Our method is based on the formation of supported lipid bilayers from cell membrane material. Using total internal reflection fluorescence microscopy, we report the contribution of viral and cellular components to the interaction kinetics of herpes simplex virus type 1 with the cell membrane. Deletion of glycoprotein C (gC), the main viral attachment glycoprotein, or deletion of heparan sulfate, an attachment factor on the cell membrane, leads to an overall decrease in association of virions to the membrane and faster dissociation from the membrane. In addition to this, we perform binding inhibition studies using the antiviral compound heparin to estimate its IC50 value. Finally, single particle tracking is used to characterize the diffusive behavior of the virus particles on the supported lipid bilayers. Altogether, our results promote this platform as a complement to existing bioanalytical assays, being at the interface between simplified artificial membrane models and live cell experiments.

Equilibrium-fluctuation analysis for interaction studies between natural ligands and single G protein-coupled receptors in native lipid vesicles.

G protein-coupled receptors (GPCRs) constitute the most versatile family of cell-membrane receptors and have been increasingly identified as important mediators of many physiological functions. They also belong to one of the most central drug target classes, but current screening technologies are limited by the requirements of overexpression and stabilization of GPCRs. This calls for sensitivity-increased detection strategies preferably meeting single-molecule detection limits. This challenge is here addressed by employing total internal reflection fluorescence microscopy to characterize the interaction kinetics between CXCR3, a GPCR involved in inflammatory responses, and two of its chemokine ligands, CXCL10 and CXCL11. Fluorescence labeling of the lipid membrane, rather than the membrane protein itself, of GPCR-containing native vesicles, and immobilization of the corresponding ligand on the surface, enabled determination of the interaction kinetics using single-molecule equilibrium-fluctuation analysis. With a limit of detection of GPCR-containing vesicles in the low picomolar concentration regime, the results demonstrate the possibility to use inhibition in solution screening of high affinity ligands/drug candidates, which due to target-binding depletion of the inhibiting compounds is demanding using assays with more moderate detection limits.

Location-specific nanoplasmonic sensing of biomolecular binding to lipid membranes with negative curvature.

The biochemical processes of cell membranes are sensitive to the geometry of the lipid bilayer. We show how plasmonic "nanowells" provide label-free real-time analysis of molecules on membranes with detection of preferential binding at negative curvature. It is demonstrated that norovirus accumulate in invaginations due to multivalent interactions with glycosphingolipids.

Preserved transmembrane protein mobility in polymer-supported lipid bilayers derived from cell membranes.

Supported lipid bilayers (SLBs) have contributed invaluable information about the physiochemical properties of cell membranes, but their compositional simplicity often limits the level of knowledge that can be gained about the structure and function of transmembrane proteins in their native environment. Herein, we demonstrate a generic protocol for producing polymer-supported lipid bilayers on glass surfaces that contain essentially all naturally occurring cell-membrane components of a cell line while still retaining transmembrane protein mobility and activity. This was achieved by merging vesicles made from synthetic lipids (PEGylated lipids and POPC lipids) with native cell-membrane vesicles to generate hybrid vesicles which readily rupture into a continuous polymer-supported lipid bilayer. To investigate the properties of these complex hybrid SLBs and particularly the behavior of their integral membrane-proteins, we used total internal reflection fluorescence imaging to study a transmembrane protease, ß-secretase 1 (BACE1), whose ectoplasmic and cytoplasmic domains could both be specifically targeted with fluorescent reporters. By selectively probing the two different orientations of BACE1 in the resulting hybrid SLBs, the role of the PEG-cushion on transmembrane protein lateral mobility was investigated. The results reveal the necessity of having the PEGylated lipids present during vesicle adsorption to prevent immobilization of transmembrane proteins with protruding domains. The proteolytic activity of BACE1 was unadulterated by the sonication process used to merge the synthetic and native membrane vesicles; importantly it was also conserved in the SLB. The presented strategy could thus serve both fundamental studies of membrane biophysics and the production of surface-based bioanalytical sensor platforms.

Coupling supported lipid bilayer electrophoresis with matrix-assisted laser desorption/ionization-mass spectrometry imaging.

Herein, we describe a new analytical platform utilizing advances in heterogeneous supported lipid bilayer (SLB) electrophoresis and matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) imaging. This platform allowed for the separation and visualization of both charged and neutral lipid membrane components without the need for extrinsic labels. A heterogeneous SLB was created using vesicles containing monosialoganglioside GM1, disialoganglioside GD1b, POPC, as well as the ortho and para isomers of Texas Red-DHPE. These components were then separated electrophoretically into five resolved bands. This represents the most complex separation by SLB electrophoresis performed to date. The SLB samples were flash frozen in liquid ethane and dried under vacuum before imaging with MALDI-MS. Fluorescence microscopy was employed to confirm the position of the Texas Red labeled lipids, which agreed well with the MALDI-MS imaging results. These results clearly demonstrate this platform's ability to isolate and identify nonlabeled membrane components within an SLB.

Phosphatidylserine reversibly binds Cu2+ with extremely high affinity.

Phosphatidylserine (PS) embedded within supported lipid bilayers was found to bind Cu(2+) from solution with extraordinarily high affinity. In fact, the equilibrium dissociation constant was in the femtomolar range. The resulting complex formed in a 1:2 Cu(2+)-to-PS ratio and quenches a broad spectrum of lipid-bound fluorophores in a reversible and pH-dependent fashion. At acidic pH values, the fluorophores were almost completely unquenched, while at basic pH values significant quenching (85-90%) was observed. The pH at which the transition occurred was dependent on the PS concentration and ranged from approximately pH 5 to 8. The quenching kinetics was slow at low Cu(2+) concentrations and basic pH values (up to several hours), while the unquenching reaction was orders of magnitude more rapid upon lowering the pH. This was consistent with diffusion-limited complex formation at basic pH but rapid dissociation under acidic conditions. The tight binding of Cu(2+) to PS may have physiological consequences under certain circumstances.

Protein separation by electrophoretic-electroosmotic focusing on supported lipid bilayers.

An electrophoretic-electroosmotic focusing (EEF) method was developed and used to separate membrane-bound proteins and charged lipids based on their charge-to-size ratio from an initially homogeneous mixture. EEF uses opposing electrophoretic and electroosmotic forces to focus and separate proteins and lipids into narrow bands on supported lipid bilayers (SLBs). Membrane-associated species were focused into specific positions within the SLB in a highly repeatable fashion. The steady-state focusing positions of the proteins could be predicted and controlled by tuning experimental conditions, such as buffer pH, ionic strength, electric field, and temperature. Careful tuning of the variables should enable one to separate mixtures of membrane proteins with only subtle differences. The EEF technique was found to be an effective way to separate protein mixtures with low initial concentrations, and it overcame diffusive peak broadening to allow four bands to be separated simultaneously within a 380 µm wide isolated supported membrane patch.

Supported bilayer electrophoresis under controlled buffer conditions.

A pH controlled flow cell device was constructed to allow electrophoretic movement of charged lipids and membrane associated proteins in supported phospholipid bilayers. The device isolated electrolysis products near the electrodes from the electrophoresis process within the bilayer. This allowed the pH over the bilayer region to remain within ±0.2 pH units or better over many hours at salt concentrations up to 10 mM. Using this setup, it was found that the electrophoretic mobility of a dye conjugated lipid (Texas Red 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (TR-DHPE)) was essentially constant between pH 3.3 and 9.3. In contrast, streptavidin, which was bound to biotinylated lipids, shifted from migrating cathodically at acidic pH values to migrating anodically under basic conditions. This shift was due to the modulation of the net charge on the protein, which changed the electrophoretic forces experienced by the macromolecule. The addition of a polyethylene glycol (PEG) cushion beneath the bilayer or the increase in the ionic strength of the buffer solution resulted in a decrease of the electroosmotic force experienced by the streptavidin with little effect on the Texas Red-DHPE. As such, it was possible in part to control the electrophoretic and electroosmotic contributions to streptavidin independently of one another.