Membrane nanotubes transform into double-membrane sheets at condensate droplets.

Cellular membranes exhibit a multitude of highly curved morphologies such as buds, nanotubes, cisterna-like sheets defining the outlines of organelles. Here, we mimic cell compartmentation using an aqueous two-phase system of dextran and poly(ethylene glycol) encapsulated in giant vesicles. Upon osmotic deflation, the vesicle membrane forms nanotubes, which undergo surprising morphological transformations at the liquid-liquid interfaces inside the vesicles. At these interfaces, the nanotubes transform into cisterna-like double-membrane sheets (DMS) connected to the mother vesicle via short membrane necks. Using super-resolution (stimulated emission depletion) microscopy and theoretical considerations, we construct a morphology diagram predicting the tube-to-sheet transformation, which is driven by a decrease in the free energy. Nanotube knots can prohibit the tube-to-sheet transformation by blocking water influx into the tubes. Because both nanotubes and DMSs are frequently formed by cellular membranes, understanding the formation and transformation between these membrane morphologies provides insight into the origin and evolution of cellular organelles.

Multispherical shapes of vesicles with intramembrane domains.

Phase separation of biomembranes into two fluid phases, a and b, leads to the formation of vesicles with intramembrane a- and b-domains. These vesicles can attain multispherical shapes consisting of several spheres connected by closed membrane necks. Here, we study the morphological complexity of these multispheres using the theory of curvature elasticity. Vesicles with two domains form two-sphere shapes, consisting of one a- and one b-sphere, connected by a closed ab-neck. The necks' effective mean curvature is used to distinguish positive from negative necks. Two-sphere shapes of two-domain vesicles can attain four different morphologies that are governed by two different stability conditions. The closed ab-necks are compressed by constriction forces which induce neck fission and vesicle division for large line tensions and/or large spontaneous curvatures. Multispherical shapes with one ab-neck and additional aa- and bb-necks involve several stability conditions, which act to reduce the stability regimes of the multispheres. Furthermore, vesicles with more than two domains form multispheres with more than one ab-neck. The multispherical shapes described here represent generalized constant-mean-curvature surfaces with up to four constant mean curvatures. These shapes are accessible to experimental studies using available methods for giant vesicles prepared from ternary lipid mixtures.

Probing the elastic response of lipid bilayers and nanovesicles to leaflet tensions via volume per lipid.

Biological and biomimetic membranes are based on lipid bilayers, consisting of two monolayers or leaflets. One important but challenging physical parameter of these membranes is their tension. For a long time, this tension was explicitly or implicitly taken to be the bilayer tension, acting on the whole bilayer membrane. More recently, it has been realized that it is useful to decompose the bilayer tension into two leaflet tensions and that these tensions are accessible to molecular dynamics simulations. To divide the bilayer up into two leaflets, it is necessary to introduce a midsurface that defines the spatial extent of the two leaflets. In previous studies, this midsurface was obtained from the density profiles across the bilayer and was then used to compute the molecular area per lipid. Here, we develop an alternative approach based on three-dimensional Voronoi tessellation and molecular volume per lipid. Using this volume-based approach, we determine the reference states with tensionless leaflets as well as the optimal volumes and areas per lipid. The optimal lipid volumes have practically the same value in both leaflets, irrespective of the size and curvature of the nanovesicles, whereas the optimal lipid areas are different for the two leaflets and depend on the vesicle size. In addition, we introduce lateral volume compressibilities to describe the elastic response of the lipid volume to the leaflet tensions. We show that the outer leaflet of a nanovesicle is more densely packed and less compressible than the inner leaflet and that this difference becomes more pronounced for smaller vesicles.

Computational analysis of protein synthesis, diffusion, and binding in compartmental biochips.

Protein complex assembly facilitates the combination of individual protein subunits into functional entities, and thus plays a crucial role in biology and biotechnology. Recently, we developed quasi-twodimensional, silicon-based compartmental biochips that are designed to study and administer the synthesis and assembly of protein complexes. At these biochips, individual protein subunits are synthesized from locally confined high-density DNA brushes and are captured on the chip surface by molecular traps. Here, we investigate single-gene versions of our quasi-twodimensional synthesis systems and introduce the trap-binding efficiency to characterize their performance. We show by mathematical and computational modeling how a finite trap density determines the dynamics of protein-trap binding and identify three distinct regimes of the trap-binding efficiency. We systematically study how protein-trap binding is governed by the system's three key parameters, which are the synthesis rate, the diffusion constant and the trap-binding affinity of the expressed protein. In addition, we describe how spatially differential patterns of traps modulate the protein-trap binding dynamics. In this way, we extend the theoretical knowledge base for synthesis, diffusion, and binding in compartmental systems, which helps to achieve better control of directed molecular self-assembly required for the fabrication of nanomachines for synthetic biology applications or nanotechnological purposes.

The importance of side branches of glycosylphosphatidylinositol anchors: a molecular dynamics perspective.

Many proteins are anchored to the cell surface of eukaryotes using a unique family of glycolipids called glycosylphosphatidylinositol (GPI) anchors. These glycolipids also exist without a covalently bound protein, in particular on the cell surfaces of protozoan parasites where they are densely populated. GPIs and GPI-anchored proteins participate in multiple cellular processes such as signal transduction, cell adhesion, protein trafficking and pathogenesis of Malaria, Toxoplasmosis, Trypanosomiasis and prion diseases, among others. All GPIs share a common conserved glycan core modified in a cell-dependent manner with additional side glycans or phosphoethanolamine residues. Here, we use atomistic molecular dynamic simulations and perform a systematic study to evaluate the structural properties of GPIs with different side chains inserted in lipid bilayers. Our results show a flop-down orientation of GPIs with respect to the membrane surface and the presentation of the side chain residues to the solvent. This finding agrees well with experiments showing the role of the side residues as active epitopes for recognition of GPIs by macrophages and induction of GPI-glycan-specific immune responses. Protein-GPI interactions were investigated by attaching parasitic GPIs to Green Fluorescent Protein. GPIs are observed to recline on the membrane surface and pull down the attached protein close to the membrane facilitating mutual contacts between protein, GPI and the lipid bilayer. This model is efficient in evaluating the interaction of GPIs and GPI-anchored proteins with membranes and can be extended to study other parasitic GPIs and proteins and develop GPI-based immunoprophylaxis to treat infectious diseases.

Large stress asymmetries of lipid bilayers and nanovesicles generate lipid flip-flops and bilayer instabilities.

Much effort has been devoted to lipid bilayers and nanovesicles with a compositional asymmetry between the two leaflets of the bilayer membranes. Here, we address another fundamental asymmetry related to lipid densities and membrane tensions. To avoid membrane rupture, the osmotic conditions must be adjusted in such a way that the bilayer membranes are subject to a relatively low bilayer tension. However, even for vanishing bilayer tension, the individual leaflets can still experience significant leaflet tensions if one leaflet is stretched whereas the other leaflet is compressed. Such a stress asymmetry between the two leaflets can be directly controlled in molecular dynamics simulations by the initial assembly of the lipid bilayers. This stress asymmetry is varied here over a wide range to determine the stability and instability regimes of the asymmetric bilayers. The stability regime shrinks with decreasing size and increasing membrane curvature of the nanovesicle. In the instability regimes, the lipids undergo stress-induced flip-flops with a flip-flop rate that increases with increasing stress asymmetry. The onset of flip-flops can be characterized by a cumulative distribution function that is well-fitted by an exponential function for planar bilayers but has a sigmoidal shape for nanovesicles. In addition, the bilayer membranes form transient non-bilayer structures that relax back towards ordered bilayers with a reduced stress asymmetry. Our study reveals intrinsic limits for the possible magnitude of the transbilayer stress asymmetry and shows that the leaflet tensions represent key parameters for the flip-flop rates.

Binding of His-tagged fluorophores to lipid bilayers of giant vesicles.

His-tagged molecules can be attached to lipid bilayers via certain anchor lipids, a method that has been widely used for the biofunctionalization of membranes and vesicles. To observe the membrane-bound molecules, it is useful to consider His-tagged molecules that are fluorescent as well. Here, we study two such molecules, green fluorescence protein (GFP) and green-fluorescent fluorescein isothiocyanate (FITC), both of which are tagged with a chain of six histidines (6H) that bind to the anchor lipids within the bilayers. The His-tag 6H is much smaller than the GFP molecule but somewhat larger than the FITC dye. The lipid bilayers form giant unilamellar vesicles (GUVs), the behavior of which can be directly observed in the optical microscope. We apply and compare three well-established preparation methods for GUVs: electroformation on platinum wire, polyvinyl alcohol (PVA) hydrogel swelling, and electroformation on indium tin oxide (ITO) glass. Microfluidics is used to expose the GUVs to a constant fluorophore concentration in the exterior solution. The brightness of membrane-bound 6H-GFP exceeds the brightness of membrane-bound 6H-FITC, in contrast to the quantum yields of the two fluorophores in solution. In fact, 6H-FITC is observed to be strongly quenched by the anchor lipids which bind the fluorophores via Ni2+ ions. For both 6H-GFP and 6H-FITC, the membrane fluorescence is measured as a function of the fluorophores' molar concentration. The theoretical analysis of these data leads to the equilibrium dissociation constants Kd = 37.5 nM for 6H-GFP and Kd = 18.5 nM for 6H-FITC. We also observe a strong pH-dependence of the membrane fluorescence.

Introduction to remodeling of biomembranes.

In general, biomembranes and giant vesicles can respond to cues in their aqueous environment by remodeling their molecular composition, shape, or topology. This themed collection focuses on remodeling of membrane shape which is intimately related to membrane curvature. In this introductory contribution, we clarify the different notions of curvature and describe the general nanoscopic mechanisms for curvature generation and membrane scaffolding. At the end, we give a brief outlook on membrane tension.

Active shape oscillations of giant vesicles with cyclic closure and opening of membrane necks.

Reaction-diffusion systems encapsulated within giant unilamellar vesicles (GUVs) can lead to shape oscillations of these vesicles as recently observed for the bacterial Min protein system. This system contains two Min proteins, MinD and MinE, which periodically attach to and detach from the GUV membranes, with the detachment being driven by ATP hydrolysis. Here, we address these shape oscillations within the theoretical framework of curvature elasticity and show that they can be understood in terms of a spontaneous curvature that changes periodically with time. We focus on the simplest case provided by a attachment-detachment kinetics that is laterally uniform along the membrane. During each oscillation cycle, the vesicle shape is transformed from a symmetric dumbbell with two subcompartments of equal size to an asymmetric dumbbell with two subcompartments of different size, followed by the reverse, symmetry-restoring transformation. This sequence of shapes is first analyzed within the spontaneous curvature model which is then extended to the area-difference-elasticity model by decomposing the spontaneous curvature into a local and nonlocal component. For both symmetric and asymmetric dumbbells, the two subcompartments are connected by a narrow membrane neck with a circular waistline. The radius of this waistline undergoes periodic oscillations, the time dependence of which can be reasonably well fitted by a single Fourier mode with an average time period of 56 s.

The glycolipid GM1 reshapes asymmetric biomembranes and giant vesicles by curvature generation.

The ganglioside GM1 is present in neuronal membranes at elevated concentrations with an asymmetric spatial distribution. It is known to generate curvature and can be expected to strongly influence the neuron morphology. To elucidate these effects, we prepared giant vesicles with GM1 predominantly present in one leaflet of the membrane, mimicking the asymmetric GM1 distribution in neuronal membranes. Based on pulling inward and outward tubes, we developed a technique that allowed the direct measurement of the membrane spontaneous curvature. Using vesicle electroporation and fluorescence intensity analysis, we were able to quantify the GM1 asymmetry across the membrane and to subsequently estimate the local curvature generated by the molecule in the bilayer. Molecular-dynamics simulations confirm the experimentally determined dependence of the membrane spontaneous curvature as a function of GM1 asymmetry. GM1 plays a crucial role in connection with receptor proteins. Our results on curvature generation of GM1 point to an additional important role of this ganglioside, namely in shaping neuronal membranes.

Collective Force Generation by Molecular Motors Is Determined by Strain-Induced Unbinding.

In the living cell, we encounter a large variety of motile processes such as organelle transport and cytoskeleton remodeling. These processes are driven by motor proteins that generate force by transducing chemical free energy into mechanical work. In many cases, the molecular motors work in teams to collectively generate larger forces. Recent optical trapping experiments on small teams of cytoskeletal motors indicated that the collectively generated force increases with the size of the motor team but that this increase depends on the motor type and on whether the motors are studied in vitro or in vivo. Here, we use the theory of stochastic processes to describe the motion of N motors in a stationary optical trap and to compute the N-dependence of the collectively generated forces. We consider six distinct motor types, two kinesins, two dyneins, and two myosins. We show that the force increases always linearly with N but with a prefactor that depends on the performance of the single motor. Surprisingly, this prefactor increases for weaker motors with a lower stall force. This counter-intuitive behavior reflects the increased probability with which stronger motors detach from the filament during strain generation. Our theoretical results are in quantitative agreement with experimental data on small teams of kinesin-1 motors.

Mechanical Tension of Biomembranes Can Be Measured by Super Resolution (STED) Microscopy of Force-Induced Nanotubes.

Membrane tension modulates the morphology of plasma-membrane tubular protrusions in cells but is difficult to measure. Here, we propose to use microscopy imaging to assess the membrane tension. We report direct measurement of membrane nanotube diameters with unprecedented resolution using stimulated emission depletion (STED) microscopy. For this purpose, we integrated an optical tweezers setup in a commercial microscope equipped for STED imaging and established micropipette aspiration of giant vesicles. Membrane nanotubes were pulled from the vesicles at specific membrane tension imposed by the aspiration pipet. Tube diameters calculated from the applied tension using the membrane curvature elasticity model are in excellent agreement with data measured directly with STED. Our approach can be extended to cellular membranes and will then allow us to estimate the mechanical membrane tension within the force-induced nanotubes.

Membrane fluctuations and acidosis regulate cooperative binding of 'marker of self' protein CD47 with the macrophage checkpoint receptor SIRPα.

Cell-cell interactions that result from membrane proteins binding weakly in trans can cause accumulations in cis that suggest cooperativity and thereby an acute sensitivity to environmental factors. The ubiquitous 'marker of self' protein CD47 binds weakly to SIRPα on macrophages, which leads to accumulation of SIRPα (also known as SHPS-1, CD172A and SIRPA) at phagocytic synapses and ultimately to inhibition of engulfment of 'self' cells - including cancer cells. We reconstituted this macrophage checkpoint with GFP-tagged CD47 on giant vesicles generated from plasma membranes and then imaged vesicles adhering to SIRPα immobilized on a surface. CD47 diffusion is impeded near the surface, and the binding-unbinding events reveal cooperative interactions as a concentration-dependent two-dimensional affinity. Membrane fluctuations out-of-plane link cooperativity to membrane flexibility with suppressed fluctuations in the vicinity of bound complexes. Slight acidity (pH 6) stiffens membranes, diminishes cooperative interactions and also reduces 'self' signaling of cancer cells in phagocytosis. Sensitivity of cell-cell interactions to microenvironmental factors - such as the acidity of tumors and other diseased or inflamed sites - can thus arise from the collective cooperative properties of flexible membranes.This article has an associated First Person interview with the first author of the paper.

Unfolding mechanism and free energy landscape of single, stable, alpha helices at low pull speeds.

Single alpha helices (SAHs) stable in isolated form are often found in motor proteins where they bridge functional domains. Understanding the mechanical response of SAHs is thus critical to understand their function. The quasi-static force-extension relation of a small number of SAHs is known from single-molecule experiments. Unknown, or still controversial, are the molecular scale details behind those observations. We show that the deformation mechanism of SAHs pulled from the termini at pull speeds approaching the quasi-static limit differs from that of typical helices found in proteins, which are stable only when interacting with other protein domains. Using molecular dynamics simulations with atomistic resolution at low pull speeds previously inaccessible to simulation, we show that SAHs start unfolding from the termini at all pull speeds we investigated. Unfolding proceeds residue-by-residue and hydrogen bond breaking is not the main event determining the barrier to unfolding. We use the molecular simulation data to test the cooperative sticky chain model. This model yields excellent fits of the force-extension curves and quantifies the distance, xE = 0.13 nm, to the transition state, the natural frequency of bond vibration, ν0 = 0.82 ns-1, and the height, V0 = 2.9 kcal mol-1, of the free energy barrier associated with the deformation of single residues. Our results demonstrate that the sticky chain model could advantageously be used to analyze experimental force-extension curves of SAHs and other biopolymers.

Simple sugars shape giant vesicles into multispheres with many membrane necks.

Simple sugars such as glucose and sucrose are ubiquitous in all organisms. One remarkable property of these small solutes is their ability to protect biomembranes against dehydration damage. This property, which reflects the underlying sugar-lipid interactions, has been intensely studied for lipid bilayers interacting with a single sugar at low hydration. Here, we use giant vesicles to investigate fully hydrated lipid membranes in contact with two sugars, glucose and sucrose. The vesicles were osmotically balanced, with the same total sugar concentration in the interior and exterior aqueous solutions. However, the two solutions differed in their composition: the interior solution contained only sucrose whereas the exterior one contained primarily glucose. This sugar asymmetry generated a striking variety of multispherical or "multi-balloon" vesicle shapes. Each multisphere involved only a single membrane that formed several spherical segments, which were connected by narrow, hourglass-shaped membrane necks. These morphologies revealed that the sugar-lipid interactions generated a significant spontaneous curvature with a magnitude of about 1 µm-1. Such a spontaneous curvature can be generated both by depletion and by adsorption layers of the sugar molecules arising from effectively repulsive and attractive sugar-lipid interactions. All multispherical shapes are stable over a wide range of parameters, with a substantial overlap between the different stability regimes, reflecting the rugged free energy landscape in shape space. One challenge for future studies is to identify pathways within this landscape that allow us to open and close the membrane necks of these shapes in a controlled and reliable manner. We will then be able to apply these multispheres as metamorphic chambers for chemical reactions and nanoparticle growth.

Decomposition of time-dependent fluorescence signals reveals codon-specific kinetics of protein synthesis.

During protein synthesis, the nascent peptide chain traverses the peptide exit tunnel of the ribosome. We monitor the co-translational movement of the nascent peptide using a fluorescent probe attached to the N-terminus of the nascent chain. Due to fluorophore quenching, the time-dependent fluorescence signal emitted by an individual peptide is determined by co-translational events, such as secondary structure formation and peptide-tunnel interactions. To obtain information on these individual events, the measured ensemble fluorescence signal has to be decomposed into position-dependent intensities. Here, we describe mRNA translation as a Markov process with specific fluorescence intensities assigned to the different states of the process. Combining the computed stochastic time evolution of the translation process with a sequence of observed ensemble fluorescence time courses, we compute the unknown position-specific intensities and obtain detailed information on the kinetics of the translation process. In particular, we find that translation of poly(U) mRNAs dramatically slows down at the fourth UUU codon. The method presented here detects subtle differences in the position-specific fluorescence intensities and thus provides a novel approach to study translation kinetics in ensemble experiments.

Bilayer Membranes with Frequent Flip-Flops Have Tensionless Leaflets.

Biomembranes are built up from lipid bilayers with two leaflets that typically differ in their lipid composition. Each lipid molecule stays within one leaflet of the bilayer before it undergoes a transition, or flip-flop, to the other leaflet. The corresponding flip-flop times are very different for different lipid species and vary over several orders of magnitude. Here, we use molecular dynamics simulations to elucidate the consequences of this separation of time scales for compositionally asymmetric bilayers. We first study bilayers with two lipid components that do not undergo flip-flops on the accessible time scales. In such a situation, one must distinguish a bilayer state in which both leaflets have the same preferred area from another state in which each leaflet is tensionless. However, when we add a third lipid component that undergoes frequent flip-flops, the bilayer relaxes toward the state with tensionless leaflets, not to the state with equal preferred leaflet areas. Furthermore, we show that bilayers with compositional asymmetry acquire a significant spontaneous curvature even if both leaflets are tensionless. Our results can be extended to lipid bilayers with a large number of lipid components provided at least one of these components undergoes frequent flip-flops. For cellular membranes containing lipid pumps, the leaflet tensions also depend on the rates of protein-induced flip-flops.

Force-Dependent Unbinding Rate of Molecular Motors from Stationary Optical Trap Data.

Molecular motors walk along filaments until they detach stochastically with a force-dependent unbinding rate. Here, we show how this unbinding rate can be obtained from the analysis of experimental data of molecular motors moving in stationary optical traps. Two complementary methods are presented, based on the analysis of the distribution for the unbinding forces and of the motor's force traces. In the first method, analytically derived force distributions for slip bonds, slip-ideal bonds, and catch bonds are used to fit the cumulative distributions of the unbinding forces. The second method is based on the statistical analysis of the observed force traces. We validate both methods with stochastic simulations and apply them to experimental data for kinesin-1.

Spherical Nanovesicles Transform into a Multitude of Nonspherical Shapes.

Nanovesicles are closed, bubblelike surfaces with a diameter between 20 and 200 nm, formed by lipid bilayers and biomembranes. Electron microscopy (EM) studies have shown that these vesicles can attain both spherical and nonspherical shapes. One disadvantage of EM methods is that they provide only a single snapshot of each vesicle. Here, we use molecular dynamics simulations to monitor the morphological transformations of individual nanovesicles. We start with the assembly of spherical vesicles that enclose a certain volume of water and contain a certain total number of lipids. When we reduce their volume, the spherical vesicles are observed to transform into a multitude of nonspherical shapes such as oblates and stomatocytes as well as prolates and dumbbells. This surprising polymorphism can be controlled by redistributing a small fraction of lipids between the inner and outer leaflets of the bilayer membranes. As a consequence, the inner and the outer leaflets experience different mechanical tensions. Small changes in the vesicle volume reduce the overall bilayer tension by 2 orders of magnitude, thereby producing tensionless bilayers. In addition, we show how to determine, for a certain total number of lipids, the unique spherical vesicle for which both leaflet tensions vanish individually. We also compute the local spontaneous curvature of the spherical membranes by identifying the first moment of the spherically symmetric stress profiles across the lipid bilayers with the nanoscopic torque as derived from curvature elasticity. Our study can be extended to other types of lipid membranes and sheds new light on cellular nanovesicles such as exosomes, which are increasingly used as biomarkers and drug delivery systems.

Sequential bottom-up assembly of mechanically stabilized synthetic cells by microfluidics.

Compartments for the spatially and temporally controlled assembly of biological processes are essential towards cellular life. Synthetic mimics of cellular compartments based on lipid-based protocells lack the mechanical and chemical stability to allow their manipulation into a complex and fully functional synthetic cell. Here, we present a high-throughput microfluidic method to generate stable, defined sized liposomes termed 'droplet-stabilized giant unilamellar vesicles (dsGUVs)'. The enhanced stability of dsGUVs enables the sequential loading of these compartments with biomolecules, namely purified transmembrane and cytoskeleton proteins by microfluidic pico-injection technology. This constitutes an experimental demonstration of a successful bottom-up assembly of a compartment with contents that would not self-assemble to full functionality when simply mixed together. Following assembly, the stabilizing oil phase and droplet shells are removed to release functional self-supporting protocells to an aqueous phase, enabling them to interact with physiologically relevant matrices.