Mechanics of biomimetic free-standing lipid membranes: insights into the elasticity of complex lipid compositions.

The creation of free-standing lipid membranes has been so far of remarkable interest to investigate processes occurring in the cell membrane since its unsupported part enables studies in which it is important to maintain cell-like physicochemical properties of the lipid bilayer, that nonetheless depend on its molecular composition. In this study, we prepare pore-spanning membranes that mimic the composition of plasma membranes and perform force spectroscopy indentation measurements to unravel mechanistic insights depending on lipid composition. We show that this approach is highly effective for studying the mechanical properties of such membranes. Furthermore, we identify a direct influence of cholesterol and sphingomyelin on the elasticity of the bilayer and adhesion between the two leaflets. Eventually, we explore the possibilities of imaging in the unsupported membrane regions. For this purpose, we investigate the adsorption and movement of a peripheral protein, the fibroblast growth factor 2, on the complex membrane.

Hydrophobin Bilayer as Water Impermeable Protein Membrane.

One of the most important properties of membranes is their permeability to water and other small molecules. A targeted change in permeability allows the passage of molecules to be controlled. Vesicles made of membranes with low water permeability are preferable for drug delivery, for example, because they are more stable and maintain the drug concentration inside. This study reports on the very low water permeability of pure protein membranes composed of a bilayer of the amphiphilic protein hydrophobin HFBI. Using a droplet interface bilayer setup, we demonstrate that HFBI bilayers are essentially impermeable to water. HFBI bilayers withstand far larger osmotic pressures than lipid membranes. Only by disturbing the packing of the proteins in the HFBI bilayer is a measurable water permeability induced. To investigate possible molecular mechanisms causing the near-zero permeability, we used all-atom molecular dynamics simulations of various HFBI bilayer models. The simulations suggest that the experimental HFBI bilayer permeability is compatible neither with a lateral honeycomb structure, as found for HFBI monolayers, nor with a residual oil layer within the bilayer or with a disordered lateral packing similar to the packing in lipid bilayers. These results suggest that the low permeabilities of HFBI and lipid bilayers rely on different mechanisms. With their extremely low but adaptable permeability and high stability, HFBI membranes could be used as an osmotic pressure-insensitive barrier in situations where lipid membranes fail such as desalination membranes.

De-wetting of evaporating drops on regular patterns of triangular posts.

Directional wicking and spreading of liquids can be achieved by regular micro-patterns of specifically designed topographic features that break the reflection symmetry of the underlying pattern. The present study aims to understand the formation and stability of wetting films during the evaporation of volatile liquid drops on surfaces with a micro-pattern of triangular posts arranged in a rectangular lattice. Depending on the density and aspect ratio of the posts, we observe either spherical-cap shaped drops with a mobile three-phase contact line or the formation of circular or angular drops with a pinned three-phase contact line. Drops of the latter class eventually evolve into a liquid film extending to the initial footprint of the drop and a shrinking cap-shaped drop sitting on the film. The drop evolution is controlled by the density and aspect ratio of the posts, while no influence of the orientation of the triangular posts on the contact line mobility becomes evident. Our experiments corroborate previous results of systematic numerical energy minimization, predicting that conditions for a spontaneous retraction of a wicking liquid film depend weakly on the orientation of the film edge relative to the micro-pattern.

Bilayer-Embedded Lipid Droplets Coated with Perilipin-2 Display a Pancake Shape.

Lipid droplets (LD) are organelles localized in the membrane of the endoplasmic reticulum (ER) that play an important role in many biological functions. Free LDs that have been released from the ER membrane and are present in the cytosol resemble an oil-in-water emulsion. The surface of an LD is coated with a phospholipid monolayer, and the core of an LD is composed of neutral lipids. Adipose differentiation-related protein (ADRP), also known as perilipin-2, is a protein that surrounds the LD, together with the phospholipid monolayer. ADRP molecules are involved in assisting in the storage of neutral lipids within LDs. In this article, we focus our interest on the influence of ADRP molecules on the 3D shape of bilayer-embedded LDs and the diffusion of phospholipids in the monolayer covering LDs. For this study, we employed two different microfluidic setups: one to produce and explore bilayer-embedded LDs and a second one to mimic the surface of a single LD. Using the first setup, we demonstrate that ADRP molecules stay preferentially localized on the surfaces of bilayer-embedded LDs, and we study their 3D-shape in the presence of ADRP. Using the second setup, we performed FRAP experiments to measure the phospholipid diffusion on a model LD surface as a function of the ADRP concentration. Although the presence of proteins on the LD surface minimally affects the phospholipid and protein motility, ADRP appears to have a significant effect on the 3D structure of LDs embedded in the bilayer.

Phospholipids diffusion on the surface of model lipid droplets.

Lipid droplets (LD) are organelles localized in the membrane of the Endoplasmic Reticulum (ER) that play an important role in metabolic functions. They consist of a core of neutral lipids surrounded by a monolayer of phosphoplipids and proteins resembling an oil-in-water emulsion droplet. Many studies have focused on the biophysical properties of these LDs. However, despite numerous efforts, we are lacking information on the mobility of phospholipids on the LDs surface, although they may play a key role in the protein distribution. In this article, we developed a microfluidic setup that allows the formation of a triolein-buffer interface decorated with a phospholipid monolayer. Using this setup, we measured the motility of phospholipid molecules by performing Fluorescent Recovery After Photobleaching (FRAP) experiments for different lipidic compositions. The results of the FRAP measurements reveal that the motility of phospholipids is controlled by the monolayer packing decorating the interface.

Cholesterol promotes clustering of PI(4,5)P2 driving unconventional secretion of FGF2.

FGF2 is a cell survival factor involved in tumor-induced angiogenesis that is secreted through an unconventional secretory pathway based upon direct protein translocation across the plasma membrane. Here, we demonstrate that both PI(4,5)P2-dependent FGF2 recruitment at the inner plasma membrane leaflet and FGF2 membrane translocation into the extracellular space are positively modulated by cholesterol in living cells. We further revealed cholesterol to enhance FGF2 binding to PI(4,5)P2-containing lipid bilayers. Based on extensive atomistic molecular dynamics (MD) simulations and membrane tension experiments, we proposed cholesterol to modulate FGF2 binding to PI(4,5)P2 by (i) increasing head group visibility of PI(4,5)P2 on the membrane surface, (ii) increasing avidity by cholesterol-induced clustering of PI(4,5)P2 molecules triggering FGF2 oligomerization, and (iii) increasing membrane tension facilitating the formation of lipidic membrane pores. Our findings have general implications for phosphoinositide-dependent protein recruitment to membranes and explain the highly selective targeting of FGF2 toward the plasma membrane, the subcellular site of FGF2 membrane translocation during unconventional secretion of FGF2.

Scaling purely elastic instability of strongly shear thinning polymer solutions.

Flow of viscoelastic polymer solutions in curved channels exhibits instability caused by the elastic nature of polymers even at low Reynolds numbers. However, scaling of the onset of this purely elastic instability in semidilute polymer solutions has not been previously reported. Here we experimentally investigate the flow of highly elastic polymer solutions above their overlap concentrations using pressure measurements and particle image velocimetry. We demonstrate that the onset of instability can be scaled by including shear dependent rheological properties of the polymer solutions in the nonlinear stability analysis. As a result, a universal criterion as function of normalized polymer concentration is provided for scaling the onset of purely elastic instability in the semidilute regime regardless of the type and molecular weight of the polymer.

Lipid Droplets Embedded in a Model Cell Membrane Create a Phospholipid Diffusion Barrier.

Lipid droplets (LDs) are ubiquitous, cytoplasmic fat storage organelles that originate from the endoplasmic reticulum (ER) membrane. They are composed of a core of neutral lipids surrounded by a phospholipid monolayer. Proteins embedded into this monolayer membrane adopt a monotopic topology and are crucial for regulated lipid storage and consumption. A key question is, which collective properties of protein-intrinsic and lipid-mediated features determine spatio-temporal protein partitioning between phospholipid bilayer and LD monolayer membranes. To address this question, a freestanding phospholipid bilayer with physiological lipidic composition is produced using microfluidics and micrometer-sized LDs are dispersed around the bilayer that spontaneously insert into the bilayer. Using confocal microscopy, the 3D geometry of the reconstituted LDs is determined with high spatial resolution. The micrometer-sized bilayer-embedded LDs present a characteristic lens shape that obeys predictions from equilibrium wetting theory. Fluorescence recovery after photobleaching measurements reveals the existence of a phospholipid diffusion barrier at the monolayer-bilayer interface. Coarse-grained molecular dynamics simulation reveals lipid specific density distributions along the pore rim, which may rationalize the diffusion barrier. The lipid diffusion barrier between the LD covering monolayer and the bilayer may be a key phenomenon influencing protein partitioning between the ER membrane and LDs in living cells.

Photoactivation of Cell-Free Expressed Archaerhodopsin-3 in a Model Cell Membrane.

Transmembrane receptor proteins are located in the plasma membranes of biological cells where they exert important functions. Archaerhodopsin (Arch) proteins belong to a class of transmembrane receptor proteins called photoreceptors that react to light. Although the light sensitivity of proteins has been intensely investigated in recent decades, the electrophysiological properties of pore-forming Archaerhodopsin (Arch), as studied in vitro, have remained largely unknown. Here, we formed unsupported bilayers between two channels of a microfluidic chip which enabled the simultaneous optical and electrical assessment of the bilayer in real time. Using a cell-free expression system, we recombinantly produced a GFP (green fluorescent protein) labelled as a variant of Arch-3. The label enabled us to follow the synthesis of Arch-3 and its incorporation into the bilayer by fluorescence microscopy when excited by blue light. Applying a green laser for excitation, we studied the electrophysiological properties of Arch-3 in the bilayer. The current signal obtained during excitation revealed distinct steps upwards and downwards, which we interpreted as the opening or closing of Arch-3 pores. From these steps, we estimated the pore radius to be 0.3 nm. In the cell-free extract, proteins can be modified simply by changing the DNA. In the future, this will enable us to study the photoelectrical properties of modified transmembrane protein constructs with ease. Our work, thus, represents a first step in studying signaling cascades in conjunction with coupled receptor proteins.

Transport Properties of Gramicidin A Ion Channel in a Free-Standing Lipid Bilayer Filled With Oil Inclusions.

Ion channels are key proteins in mammalian cell membranes. They have a central role in the physiology of excitable cells such as neurons, muscle, and heart cells. They also play a crucial role in kidney physiology. The gramicidin ion channel is one of the most studied ion channels, in particular it was intensively employed to investigate the lipid-protein interactions in model cell membranes. For example, even though the sequence of gramicidin is extremely hydrophobic, its motion is impaired in membrane bilayer, i.e., it does not rapidly flip to the other membrane leaflet, and low channel activity were observed when gramicidin is added asymmetrically to only one leaflet of a model cell membrane. In this article, we study the transport properties of gramicidin channel in a heterogeneous model membrane. Using microfluidics, we are forming freestanding bilayers as model cell membranes including heterogeneous domains that are created by oil inclusions. The presence of oil inclusions is then demonstrated by measuring the bilayer capacity via a patch-clamp amplifier and fluorescent confocal inspection. Based on electrophysiological and optical measurements Gramicidin A (gA) ion channels are dispersed into the buffer phases on both side of the formed lipid bilayer and insert spontaneously into the bilayer upon formation. The presence of functional Gramicidin A is then demonstrated by measuring conductivity signals. Based on electrophysiological and optical measurements, we explore the consequence of the presence of these oil inclusions on the functionality of incorporated gA ion channels. For low oil concentration, we measure a decrease of gA transport properties due to the reduction of the bilayer tension. For large oil concentration, we measure a saturation of gA transport properties due to an increase of the bilayer thickness.

Flowing droplet interface bilayers: A microfluidic tool to control droplet trajectories and to study mechanical properties of unsupported lipid bilayers.

We introduce the concept of Flowing Droplet Interface Bilayers (FDIBs) that are made of two droplets maintained in contact due to the presence of an adhesive lipidic surfactant. This system is similar to a flowing dumbbell made of two droplets interconnected by a lipid bilayer and driven by an external flow. Interestingly, such a dumbbell does not show a straight flow trajectory, but it oscillates between the sidewalls while moving along the microchannel. The origin of this unusual motion is hydrodynamic interactions, as demonstrated by analytical calculations and micro particle image velocimentry (µPiV) measurements. The hydrodynamic motion appears to be highly sensitive to the mechanical properties of the lipid bilayer connecting the two droplets (FDIB). Thus, droplet trajectories can be controlled by tuning the lipid bilayer composition, which enables in turn investigating mechanical properties of free-standing lipid bilayers.

Kinetics of active water/ethanol Janus droplets.

Droplets made of a water/ethanol mixture spontaneously self-propel in an oil/surfactant solution and, depending on the initial ethanol concentration at the time of production, may evolve in up to three stages. Upon self-propulsion the droplets absorb surfactant molecules during their continuous motion in the oily phase. In combination with the continuous loss of ethanol this mass exchange with the ambient phase may lead to a spontaneous phase separation of the water/ethanol mixture, and eventually to the formation of characteristic Janus droplets. Supported by experimental evidence, we propose a simple model that is able to explain the propulsion velocity and its scaling with the droplet radius in the last stage of the droplet evolution.

Simultaneous measurement of surface and bilayer tension in a microfluidic chip.

Freestanding lipid bilayers are one of the most used model systems to mimic biological cell membranes. To form an unsupported bilayer, we employ two aqueous fingers in a microfluidic chip surrounded by an oily phase that contains lipids. Upon pushing two aqueous fingers forward, their interface becomes decorated with a lipid monolayer and eventually zip to form a bilayer when the monolayers have nanoscopic contact with each other. Using this straightforward approach, the quick and easy bilayer formation is facilitated by oil draining into the microfluidic device material consisting of polydimethylsiloxane. However, the oil drainage limits the lifetime of a bilayer to about 1 h. We demonstrate that this drainage can be managed, resulting in superior bilayer stability and an increased lifetime of several hours when using a pressure-controlled system. Applying different pressures to the aqueous fingers in the microfluidic chip, the formed bilayer can even be bent to a desired curvature. Extracting the contact angle and the resulting curvature of the bilayer region, for a given applied pressure difference, both the bilayer tension and the surface tension of each lipid monolayer can be derived from a single experiment using the Young Laplace pressure equation.

Directional Liquid Wicking in Regular Arrays of Triangular Posts.

Wicking of wetting liquids into micropatterns of posts with homogeneous triangular cross section is studied in experiments and by numerical energy minimizations. To test for directional wicking, we fabricated regular arrays of posts with various combinations of line fractions and aspect ratios using standard photolithography processes. In agreement with numerical energy minimizations of the liquid film morphology, we find spontaneous wicking in the experiments only for line fractions and aspect ratios where the homogeneous liquid film represents the state of lowest interfacial free energy and where no local energy minimum could be detected in our numerical energy minimizations. The numerical results further demonstrate that the stability of a certain morphology of the terminal meniscus controls the direction of wicking relative to the orientation of the triangular posts. The observed selectivity of spontaneous wicking with respect to the meniscus orientation can be exploited to build a microfluidic rectifier for partially wetting liquids.

Tension-Induced Translocation of an Ultrashort Carbon Nanotube through a Phospholipid Bilayer.

Increasing awareness of bioeffects and toxicity of nanomaterials interacting with cells puts in focus the mechanisms by which nanomaterials can cross lipid membranes. Apart from well-discussed energy-dependent endocytosis for large objects and passive diffusion through membranes by solute molecules, other translocation mechanisms based on physical principles can exist. We show the importance of membrane tension on the translocation through lipid bilayers of ultrashort carbon nanotubes (USCNTs). By using a combination of a microfluidic setup and single chain mean field (SCMF) theory, we observed that, under membrane tension, USCNT inserted into a lipid bilayer may spontaneously nucleate an unstable local pore, allowing it to escape from the bilayer. We demonstrated that stretching of the membrane is essential for triggering this mechanism of translocation, and no translocation is observed at low membrane tension. For this purpose, a quantitative analysis of the kinetic pathway associated with USCNT translocation induced by tension was performed in a specially designed microfluidic device, simultaneously combining optical fluorescence microscopy and electrophysiological measurements. An important outcome of these findings is the identification of the way to control the nanomaterial translocation through the lipid bilayer by membrane tension that can be useful in many practical applications.

Impact of energy dissipation on interface shapes and on rates for dewetting from liquid substrates.

We revisit the fundamental problem of liquid-liquid dewetting and perform a detailed comparison of theoretical predictions based on thin-film models with experimental measurements obtained by atomic force microscopy. Specifically, we consider the dewetting of a liquid polystyrene layer from a liquid polymethyl methacrylate layer, where the thicknesses and the viscosities of both layers are similar. Using experimentally determined system parameters like viscosity and surface tension, an excellent agreement of experimentally and theoretically obtained rim profile shapes are obtained including the liquid-liquid interface and even dewetting rates. Our new energetic approach additionally allows to assess the physical importance of different contributions to the energy-dissipation mechanism, for which we analyze the local flow fields and the local dissipation rates. Using this approach, we explain why dewetting rates for liquid-liquid systems follow no universal power law, despite the fact that experimental velocities are almost constant. This is in contrast to dewetting scenarios on solid substrates and in contrast to previous results for liquid-liquid substrates using heuristic approaches.

Shape Evolution of Droplets Growing on Linear Microgrooves.

Anisotropic spreading of liquids and elongated droplet shapes are often encountered on surfaces decorated with a periodic micropattern of linear surface topographies. Numerical calculations and wetting experiments show that the shape evolution of droplets that are slowly growing on a surface with parallel grooves can be grouped into two distinct morphological regimes. In the first regime, the liquid of the growing droplet spreads only into the direction parallel to the grooves. In the second regime, the three-phase contact line advances also perpendicular to the grooves, whereas the growing droplets approach a scale-invariant shape. Here, we demonstrate that shapes of droplets in contact with a large number of linear grooves are identical to the shapes of droplets confined to a plane chemical stripe, where this mapping of shapes is solely based on the knowledge of the cross section of the linear grooves and the material contact angle. The spectrum of interfacial shapes on the chemical stripe can be exploited to predict the particular growth mode and the asymptotic value of the base eccentricity in the limit of droplets covering a large number of grooves. The proposed model shows an excellent agreement with experimentally observed base eccentricities for droplets on grooves of various cross sections. The universality of the model is underlined by the accurate match with available literature data for droplet eccentricities on parallel chemical stripes.

Adhesion Properties of Freestanding Hydrophobin Bilayers.

Hydrophobins are a family of small-sized proteins featuring a distinct hydrophobic patch on the protein's surface, rendering them amphiphilic. This particularity allows hydrophobins to self-assemble into monolayers at any hydrophilic/hydrophobic interface. Moreover, stable pure protein bilayers can be created from two interfacial hydrophobin monolayers by contacting either their hydrophobic or their hydrophilic sides. In this study, this is achieved via a microfluidic approach, in which also the bilayers' adhesion energy can be determined. This enables us to study the origin of the adhesion of hydrophobic and hydrophilic core bilayers made from the class II hydrophobins HFBI and HFBII. Using different fluid media in this setup and introducing genetically modified variants of the HFBI molecule, the different force contributions to the adhesion of the bilayer sheets are studied. It was found that in the hydrophilic contact situation, the adhesive interaction was higher than that in the hydrophobic contact situation and could be even enhanced by reducing the contributions of electrostatic interactions. This effect indicates that the van der Waals interaction is the dominant contribution that explains the stability of the observed bilayers.

Apatite nanoparticles strongly improve red blood cell cryopreservation by mediating trehalose delivery via enhanced membrane permeation.

Cryopreservation of red blood cells (RBC) is an important method for maintaining an inventory of rare RBC units and managing special transfusion circumstances. Currently, in a clinical setting, glycerol is used as cryoprotectant against freezing damage. After thawing and before transfusion, glycerol must however be removed to avoid intravascular hemolysis, via a complex and time-consuming deglycerolization process which requires specialized equipment. Improved cryopreservation methods using non-toxic agents are required to increase biocompatibility and decrease processing time. Biocompatible cryoprotectants (e.g. trehalose) were proposed, but their low permeation through RBC membranes limits their cryoprotection efficacy. Herein, we report for the first time a glycerol-free cryopreservation approach, using colloidal bioinspired apatite nanoparticles (NP) as bioactive promoters of RBC cryopreservation mediated by trehalose. Addition of apatite NP in the medium tremendously increases RBC cryosurvival, up to 91% (42% improvement compared to a control without NP) which is comparable to FDA-approved cryoprotection protocol employing glycerol. NP concentration and incubation conditions strongly modulate the NP bioactivity. Complementary experimental and computational analyses of the interaction between apatite NP and model lipid bilayers revealed complex events occurring at the NP-bilayer interface. Apatite NP do not cross the bilayer but momentarily modulate its physical status. These changes affect the membrane behavior, and promote the permeation of trehalose and a model fluorescent molecule (FITC). This approach is a new alternative to using toxic glycerol for cells cryopreservation, and the identification of this enhancing no-pore permeation mechanism of apatite NP appears as an original delivery pathway for cryoprotectant agents and beyond.

The Role of Local Instabilities in Fluid Invasion into Permeable Media.

Wettability is an important factor which controls the displacement of immiscible fluids in permeable media, with far reaching implications for storage of CO2 in deep saline aquifers, fuel cells, oil recovery, and for the remediation of oil contaminated soils. Considering the paradigmatic case of random piles of spherical beads, fluid front morphologies emerging during slow immiscible displacement are investigated in real time by X-ray micro-tomography and quantitatively compared with model predictions. Controlled by the wettability of the bead matrix two distinct displacement patterns are found. A compact front morphology emerges if the invading fluid wets the beads while a fingered morphology is found for non-wetting invading fluids, causing the residual amount of defending fluid to differ by one order of magnitude. The corresponding crossover between these two regimes in terms of the advancing contact angle is governed by an interplay of wettability and pore geometry and can be predicted on the basis of a purely quasi-static consideration of local instabilities that control the progression of the invading interface.