Microplastics destabilize lipid membranes by mechanical stretching.

Estimated millions of tons of plastic are dumped annually into oceans. Plastic has been produced only for 70 y, but the exponential rise of mass production leads to its widespread proliferation in all environments. As a consequence of their large abundance globally, microplastics are also found in many living organisms including humans. While the health impact of digested microplastics on living organisms is debatable, we reveal a physical mechanism of mechanical stretching of model cell lipid membranes induced by adsorbed micrometer-sized microplastic particles most commonly found in oceans. Combining experimental and theoretical approaches, we demonstrate that microplastic particles adsorbed on lipid membranes considerably increase membrane tension even at low particle concentrations. Each particle adsorbed at the membrane consumes surface area that is proportional to the contact area between particle and the membrane. Although lipid membranes are liquid and able to accommodate mechanical stress, the relaxation time is much slower than the rate of adsorption; thus, the cumulative effect from arriving microplastic particles to the membrane leads to the global reduction of the membrane area and increase of membrane tension. This, in turn, leads to a strong reduction of membrane lifetime. The effect of mechanical stretching of microplastics on living cells membranes was demonstrated by using the aspiration micropipette technique on red blood cells. The described mechanical stretching mechanism on lipid bilayers may provide better understanding of the impact of microplastic particles in living systems.

Nascent fusion pore opening monitored at single-SNAREpin resolution.

Vesicle fusion with a target membrane is a key event in cellular trafficking and ensures cargo transport within the cell and between cells. The formation of a protein complex, called SNAREpin, provides the energy necessary for the fusion process. In a three-dimensional microfluidic chip, we monitored the fusion of small vesicles with a suspended asymmetric lipid bilayer. Adding ion channels into the vesicles, our setup allows the observation of a single fusion event by electrophysiology with 10-µs precision. Intriguingly, we identified that small transient fusion pores of discrete sizes reversibly opened with a characteristic lifetime of ∼350 ms. The distribution of their apparent diameters displayed two peaks, at 0.4 ± 0.1 nm and 0.8 ± 0.2 nm. Varying the number of SNAREpins, we demonstrated that the first peak corresponds to fusion pores induced by a single SNAREpin and the second peak is associated with pores involving two SNAREpins acting simultaneously. The pore size fluctuations provide a direct estimate of the energy landscape of the pore. By extrapolation, the energy landscape for three SNAREpins does not exhibit any thermally significant energy barrier, showing that pores larger than 1.5 nm are spontaneously produced by three or more SNAREpins acting simultaneously, and expand indefinitely. Our results quantitatively explain why one SNAREpin is sufficient to open a fusion pore and more than three SNAREpins are required for cargo release. Finally, they also explain why a machinery that synchronizes three SNAREpins, or more, is mandatory to ensure fast neurotransmitter release during synaptic transmission.

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.

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.

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.

Unexpected Cholesterol-Induced Destabilization of Lipid Membranes near Transmembrane Carbon Nanotubes.

Cholesterol is a crucial component of mammalian cell membranes that takes part in many vital processes. It is generally accepted that cholesterol stabilizes the membrane and induces transitions into ordered states. In contrast to expectations, we demonstrate that cholesterol can destabilize the membrane by creating a nanodomain around a perpendicularly embedded ultrashort carbon nanotube (CNT), and we show that cholesterol triggers the translocation of an ultrashort CNT through the cell membrane. Using atomistic simulations, we report the existence of a nanoscale domain around an ultrashort carbon nanotube within a crossover distance of 0.9 nm from the surface of the nanotube, where the properties of the bilayer are different from the bulk: the domain is characterized by increased fluctuations, increased thickness, and increased order of the lipids with respect to the bulk. Cholesterol decreases the thickness and order of lipids and increases the fluctuations with respect to a pure lipid bilayer. Experimentally, we confirm that cholesterol nanodomains provoke spontaneous translocation of nanotubes through a lipid bilayer even for low membrane tensions. A specially designed microfluidic device allows us to trace the kinetic pathway of the translocation process and establish the threshold cholesterol concentration of 20% for translocation. The reported nanoscale cholesterol-induced membrane restructuring near the ultrashort CNT in lipid membranes enables precise control and specific targeting of a membrane using cholesterol. As an example, it may allow for specific targeting between cholesterol-rich mammalian cells and cholesterol-poor bacterial cells.

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.

Highly Reproducible Physiological Asymmetric Membrane with Freely Diffusing Embedded Proteins in a 3D-Printed Microfluidic Setup.

Experimental setups to produce and to monitor model membranes have been successfully used for decades and brought invaluable insights into many areas of biology. However, they all have limitations that prevent the full in vitro mimicking and monitoring of most biological processes. Here, a suspended physiological bilayer-forming chip is designed from 3D-printing techniques. This chip can be simultaneously integrated to a confocal microscope and a path-clamp amplifier. It is composed of poly(dimethylsiloxane) and consists of a ≈100 µm hole, where the horizontal planar bilayer is formed, connecting two open crossed-channels, which allows for altering of each lipid monolayer separately. The bilayer, formed by the zipping of two lipid leaflets, is free-standing, horizontal, stable, fluid, solvent-free, and flat with the 14 types of physiologically relevant lipids, and the bilayer formation process is highly reproducible. Because of the two channels, asymmetric bilayers can be formed by making the two lipid leaflets of different composition. Furthermore, proteins, such as transmembrane, peripheral, and pore-forming proteins, can be added to the bilayer in controlled orientation and keep their native mobility and activity. These features allow in vitro recapitulation of membrane process close to physiological conditions.

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.

Collective waves in dense and confined microfluidic droplet arrays.

Excitation mechanisms for collective waves in confined dense one-dimensional microfluidic droplet arrays are investigated by experiments and computer simulations. We demonstrate that distinct modes can be excited by creating specific 'defect' patterns in flowing droplet trains. Excited longitudinal modes exhibit a short-lived cascade of pairs of laterally displacing droplets. Transversely excited modes obey the dispersion relation of microfluidic phonons and induce a coupling between longitudinal and transverse modes, whose origin is the hydrodynamic interaction of the droplets with the confining walls. Moreover, we investigate the long-time behaviour of the oscillations and discuss possible mechanisms for the onset of instabilities. Our findings demonstrate that the collective dynamics of microfluidic droplet ensembles can be studied particularly well in dense and confined systems. Experimentally, the ability to control microfluidic droplets may allow the modulation of the refractive index of optofluidic crystals, which is a promising approach for the production of dynamically programmable metamaterials.

Coexistence of different droplet generating instabilities: new breakup regimes of a liquid filament.

The coexistence of multiple droplet breakup instabilities in a Step-emulsification geometry is studied. A liquid filament, which is confined in one dimension by channel walls and surrounded by a co-flowing immiscible continuous phase, decays into droplets when subject to a sudden release of confinement. Depending on the filament aspect ratio and liquid flow rates, an unexpectedly rich variety of droplet breakup regimes is found. All of these breakup regimes are composed of two basic instabilities, i.e. a step- and a jet-instability, that coexist in various combinations on the same filament. Surprisingly, even an asymmetric breakup regime is found, producing droplet families of significantly different diameters, while the filament is subject to a fully symmetric flow field. We suggest key physical principles explaining the spontaneous symmetry breaking and the transitions between individual droplet breakup regimes. The particular ability to produce distinct droplet families from a single filament is demonstrated to allow for simultaneous concentration and encapsulation of particles into one droplet family while excess bulk liquid is released into another family of droplets.

Fast membrane hemifusion via dewetting between lipid bilayers.

The behavior of lipid bilayers is important to understand the functionality of cells like the trafficking of ions. Standard procedures to explore the properties of lipid bilayers and hemifused states typically use supported membranes or vesicles. Both techniques have several shortcomings in terms of bio-relevance or accessibility for measurements. In this article, the formation of individual free standing hemifused states between model cell membranes is studied using an optimized microfluidic scheme which allows for simultaneous optical and electrophysiological measurements. In the first step, two model membranes are formed at a desired location within a microfluidic device using a variation of the droplet interface bilayer (DiB) technique. In the second step, the two model membranes are brought into contact forming a single hemifused state. For all tested lipids, the hemifused state between free standing membranes forms within hundreds of milliseconds, i.e. several orders of magnitude faster than those reported in literature. The formation of a hemifused state is observed as a two stage process, whereas the second stage can be explained as a dewetting process under no-slip boundary conditions. The formed hemifusion states have a long lifetime and a single fusion event can be observed when triggered by an applied electric field as demonstrated for monoolein.

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.

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.

Protein-coated nanoparticles exhibit Lévy flights on a suspended lipid bilayer.

Lateral diffusion of nano-objects on lipid membranes is a crucial process in cell biology. Recent studies indicate that nanoparticle lateral diffusion is affected by the presence of membrane proteins and deviates from Brownian motion. Gold nanoparticles (Au NPs) stabilized by short thiol ligands were dispersed near a free-standing bilayer formed in a 3D microfluidic chip. Using dark-field microscopy, the position of single NPs at the bilayer surface was tracked over time. Numerical analysis of the NP trajectories shows that NP diffusion on the bilayer surface corresponds to Brownian motion. The addition of bovine serum albumin (BSA) protein to the solution led to the formation of a protein corona on the NP surface. We found that protein-coated NPs show anomalous superdiffusion and that the distribution of their relative displacement obeys Lévy flight statistics. This superdiffusive motion is attributed to a drastic reduction in adhesive energies between the NPs and the bilayer in the presence of the protein corona. This hypothesis was confirmed by numerical simulations mimicking the random walk of a single particle near a weakly adhesive surface. These results may be generalized to other classes of nano-objects that experience adsorption-desorption behaviour with a weakly adhesive surface.

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.

Protein corona modulates interaction of spiky nanoparticles with lipid bilayers.

The impact of protein corona on the interactions of nanoparticles (NPs) with cells remains an open question. This question is particularly relevant to NPs which sizes, ranging from tens to hundreds nanometers, are comparable to the sizes of most abundant proteins in plasma. Protein sizes match with typical thickness of various coatings and ligands layers, usually present at the surfaces of larger NPs. Such size match may affect the properties and the designed function of NPs. We offer a direct demonstration of how protein corona can dramatically change the interaction mode between NPs and lipid bilayers. To this end, we choose the most extreme case of NP surface modification: nanostructures in the form of rigid spikes of 10-20 nm length at the surface of gold nanoparticles. In the absence of proteins we observe the formation of reversible pores when spiky NPs adsorb on lipid bilayers. In contrast, the presence of bovine serum albumin (BSA) proteins adsorbed at the surface of spiked NPs, effectively reduces the length of spikes exposed to the interaction with lipid bilayers. Thus, protein corona changes qualitatively the dynamics of pore formation, which is completely suppressed at high protein concentrations. These results suggest that protein corona can not only be critical for interaction of NPs with membranes, it may change their mode of interaction, thus offsetting the role of surface chemistry and ligands.

Enhanced water permeability across a physiological droplet interface bilayer doped with fullerenes.

We measure the water permeability across a physiological lipid bilayer produced by the droplet interface bilayer (DiB) technique. This lipid bilayer can be considered as physiologically relevant because it presents a lipidic composition close to human cell membranes. The measured water permeability coefficients across this lipid bilayer are reported as a function of the cholesterol concentration. It is found that the water permeability coefficients decreased with increasing cholesterol concentration, in agreement with the existing literature. And, consistently, the extracted corresponding activation energies increase with increasing cholesterol concentration in the lipid bilayer. Hence having demonstrated the robustness of the experimental system, we extend this study by exploring the influence of fullerenes on the water permeability of a physiological lipid bilayer. Interestingly, we observe a significant increase of the measured water permeability coefficients across this lipid bilayer for large fullerenes concentration. This enhanced permeability might be related to the conductive properties of fullerenes.

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.