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.

Leaflet Tensions Control the Spatio-Temporal Remodeling of Lipid Bilayers and Nanovesicles.

Biological and biomimetic membranes are based on lipid bilayers, which consist of two monolayers or leaflets. To avoid bilayer edges, which form when the hydrophobic core of such a bilayer is exposed to the surrounding aqueous solution, a single bilayer closes up into a unilamellar vesicle, thereby separating an interior from an exterior aqueous compartment. Synthetic nanovesicles with a size below 100 nanometers, traditionally called small unilamellar vesicles, have emerged as potent platforms for the delivery of drugs and vaccines. Cellular nanovesicles of a similar size are released from almost every type of living cell. The nanovesicle morphology has been studied by electron microscopy methods but these methods are limited to a single snapshot of each vesicle. Here, we review recent results of molecular dynamics simulations, by which one can monitor and elucidate the spatio-temporal remodeling of individual bilayers and nanovesicles. We emphasize the new concept of leaflet tensions, which control the bilayers' stability and instability, the transition rates of lipid flip-flops between the two leaflets, the shape transformations of nanovesicles, the engulfment and endocytosis of condensate droplets and rigid nanoparticles, as well as nanovesicle adhesion and fusion. To actually compute the leaflet tensions, one has to determine the bilayer's midsurface, which represents the average position of the interface between the two leaflets. Two particularly useful methods to determine this midsurface are based on the density profile of the hydrophobic lipid chains and on the molecular volumes.

Different pathways for engulfment and endocytosis of liquid droplets by nanovesicles.

During endocytosis of nanoparticles by cells, the cellular membranes engulf the particles, thereby forming a closed membrane neck that subsequently undergoes fission. For solid nanoparticles, these endocytic processes have been studied in some detail. Recently, such processes have also been found for liquid and condensate droplets, both in vitro and in vivo. These processes start with the spreading of the droplet onto the membrane followed by partial or complete engulfment of the droplet. Here, we use molecular dynamics simulations to study these processes at the nanoscale, for nano-sized droplets and vesicles. For both partial and complete engulfment, we observe two different endocytic pathways. Complete engulfment leads to a closed membrane neck which may be formed in a circular or strongly non-circular manner. A closed circular neck undergoes fission, thereby generating two nested daughter vesicles whereas a non-circular neck hinders the fission process. Likewise, partial engulfment of larger droplets leads to open membrane necks which can again have a circular or non-circular shape. Two key parameters identified here for these endocytic pathways are the transbilayer stress asymmetry of the vesicle membrane and the positive or negative line tension of the membrane-droplet contact line.

Budding and Fission of Nanovesicles Induced by Membrane Adsorption of Small Solutes.

Membrane budding and fission are essential cellular processes that produce new membrane compartments during cell and organelle division, for intracellular vesicle trafficking as well as during endo- and exocytosis. Such morphological transformations have also been observed for giant lipid vesicles with a size of many micrometers. Here, we report budding and fission processes of lipid nanovesicles with a size below 50 nm. We use coarse-grained molecular dynamics simulations, by which we can visualize the morphological transformations of individual vesicles. The budding and fission processes are induced by low concentrations of small solutes that absorb onto the outer leaflets of the vesicle membranes. In addition to the solute concentration, we identify the solvent conditions as a second key parameter for these processes. For good solvent conditions, the budding of a nanovesicle can be controlled by reducing the vesicle volume for constant solute concentration or by increasing the solute concentration for constant vesicle volume. After the budding process is completed, the budded vesicle consists of two membrane subcompartments which are connected by a closed membrane neck. The budding process is reversible as we demonstrate explicitly by reopening the closed neck. For poor solvent conditions, on the other hand, we observe two unexpected morphological transformations of nanovesicles. Close to the binodal line, at which the aqueous solution undergoes phase separation, the vesicle exhibits recurrent shape changes with closed and open membrane necks, reminiscent of flickering fusion pores (kiss-and-run) as observed for synaptic vesicles. As we approach the binodal line even closer, the recurrent shape changes are truncated by the fission of the membrane neck which leads to the division of the nanovesicle into two daughter vesicles. In this way, our simulations reveal a nanoscale mechanism for the budding and fission of nanovesicles, a mechanism that arises from the interplay between membrane elasticity and solute-mediated membrane adhesion.

Mapping a single-molecule folding process onto a topological space.

Physics of protein folding has been dominated by conceptual frameworks including the nucleation-propagation mechanism and the diffusion-collision model, and none address the topological properties of a chain during a folding process. Single-molecule interrogation of folded biomolecules has enabled real-time monitoring of folding processes at an unprecedented resolution. Despite these advances, the topology landscape has not been fully mapped for any chain. Using a novel circuit topology approach, we map the topology landscape of a model polymeric chain. Inspired by single-molecule mechanical interrogation studies, we restrained the ends of a chain and followed fold nucleation dynamics. We find that, before the nucleation, transient local entropic loops dominate. Although the nucleation length of globules is dependent on the cohesive interaction, the ultimate topological states of the collapsed polymer are largely independent of the interaction but depend on the speed of the folding process. After the nucleation, transient topological rearrangements are observed that converge to a steady-state, where the fold grows in a self-similar manner.

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.

Nanodroplets at Membranes Create Tight-Lipped Membrane Necks via Negative Line Tension.

The response of biomembranes to aqueous-phase separation and to the resulting water-in-water droplets has been recently studied on the micrometer scale using optical microscopy and elasticity theory. When such a droplet adheres to the membrane, it forms a contact area that is bounded by a contact line. For a micrometer-sized droplet, the line tension associated with this contact line can usually be ignored compared with the surface tensions. However, for a small nanoscopic droplet, this line tension is expected to affect the membrane-droplet morphology. Here, we use molecular simulations to study nanodroplets at membranes and to gain insight into these line tension effects. The latter effects are shown to depend strongly on another key parameter, the mechanical tension experienced by the membrane. For a large membrane tension, a droplet adhering to the membrane is only partially engulfed by the membrane, and the membrane-droplet system exhibits an axisymmetric morphology. A reduction of the membrane tension leads to an increase in the contact area and a decrease in the interfacial area of the droplet, initially retaining its axisymmetric shape, which implies a circular contact line and a circular membrane neck. However, when the tension falls below a certain threshold value, the system undergoes a morphological transition toward a non-axisymmetric morphology with a non-circular membrane neck. This morphology persists until the nanodroplet is completely engulfed by the membrane and the membrane neck has closed into a tight-lipped shape. The latter morphology is caused by a negative line tension, which is shown to be a robust feature of membrane-droplet systems. A closed membrane neck with a tight-lipped shape suppresses both thermally activated and protein-induced scission of the neck, implying a reduction in the cellular uptake of nanodroplets by pinocytosis and fluid-phase endocytosis. Furthermore, based on our results, we can also draw important conclusions about the time-dependent processes corresponding to the surface nucleation and growth of nanodroplets at membranes.

Topology of polymer chains under nanoscale confinement.

Spatial confinement limits the conformational space accessible to biomolecules but the implications for bimolecular topology are not yet known. Folded linear biopolymers can be seen as molecular circuits formed by intramolecular contacts. The pairwise arrangement of intra-chain contacts can be categorized as parallel, series or cross, and has been identified as a topological property. Using molecular dynamics simulations, we determine the contact order distributions and topological circuits of short semi-flexible linear and ring polymer chains with a persistence length of lp under a spherical confinement of radius Rc. At low values of lp/Rc, the entropy of the linear chain leads to the formation of independent contacts along the chain and accordingly, increases the fraction of series topology with respect to other topologies. However, at high lp/Rc, the fraction of cross and parallel topologies are enhanced in the chain topological circuits with cross becoming predominant. At an intermediate confining regime, we identify a critical value of lp/Rc, at which all topological states have equal probability. Confinement thus equalizes the probability of more complex cross and parallel topologies to the level of the more simple, non-cooperative series topology. Moreover, our topology analysis reveals distinct behaviours for ring- and linear polymers under weak confinement; however, we find no difference between ring- and linear polymers under strong confinement. Under weak confinement, ring polymers adopt parallel and series topologies with equal likelihood, while linear polymers show a higher tendency for series arrangement. The radial distribution analysis of the topology reveals a non-uniform effect of confinement on the topology of polymer chains, thereby imposing more pronounced effects on the core region than on the confinement surface. Additionally, our results reveal that over a wide range of confining radii, loops arranged in parallel and cross topologies have nearly the same contact orders. Such degeneracy implies that the kinetics and transition rates between the topological states cannot be solely explained by contact order. We expect these findings to be of general importance in understanding chaperone assisted protein folding, chromosome architecture, and the evolution of molecular folds.

Topology of internally constrained polymer chains.

Linear chains with intra-chain contacts can adopt different topologies and allow transitions between them, but it remains unclear how this process can be controlled. This question is important to systems ranging from proteins to chromosomes, which can adopt different conformations that are key to their function and toxicity. Here, we investigate how the topological dynamics of a simple linear chain is affected by interactions with a binding partner, using Monte Carlo and Molecular Dynamics simulations. We show that two point contacts with a binding partner are sufficient to accelerate or decelerate the formation of particular topologies within linear chains. Computed ''folding-time landscapes" that detail the folding time within the topology space show that such contacts deform these landscapes and hence alter the occupation probability of topological states. The results provide a mechanism by which chain topologies can be controlled externally, which opens up the possibility of regulating topological dynamics and the formation of more complex topologies. The findings may have important implications for understanding the mechanism of chaperone action as well as genome architecture and evolution.

Is the Solution Activity Derivative Sufficient to Parametrize Ion-Ion Interactions? Ions for TIP5P Water.

Biomolecular processes involve hydrated ions, and thus molecular simulations of such processes require accurate force-field parameters for these ions. In the best force-fields, both ion-water and anion-cation interactions are explicitly parametrized. First, the ion Lennard-Jones parameters are optimized to reproduce, for example, single ion solvation free energies; then ion-pair interactions are adjusted to match experimental activity or activity derivatives. Here, we apply this approach to derive optimized parameters for concentrated NaCl, KCl, MgCl2, and CaCl2 salt solutions, to be used with the TIP5P water model. These parameters are of interest because of a number of desirable properties of the TIP5P water model, especially for the simulation of carbohydrates. The results show, that this state of the art approach is insufficient, because the activity derivative often reaches a plateau near the target experimental value, for a wide range of parameter values. The plateau emerges from the interconversion between different types of ion pairs, so parameters leading to equally good agreement with the target solution activity or activity derivative yield very different solution structures. To resolve this indetermination, a second target property, such as the experimentally determined ion-ion coordination number, is required to uniquely determine anion-cation interactions. Simulations show that combining activity derivatives and coordination number as experimental target properties to parametrize ion-ion interactions, is a powerful method for reliable ion-water force field parametrization, and gives insight into the concentration of contact or solvent shared ion pairs in a wide range of salt concentrations. For the alkali and halide ions Li+, Rb+, Cs+, F-, Br-, and I-, we present ion-water parameters appropriate at infinite dilution only.

Cell-imprinted substrates act as an artificial niche for skin regeneration.

Bioinspired materials can mimic the stem cell environment and modulate stem cell differentiation and proliferation. In this study, biomimetic micro/nanoenvironments were fabricated by cell-imprinted substrates based on mature human keratinocyte morphological templates. The data obtained from atomic force microscopy and field emission scanning electron microscopy revealed that the keratinocyte-cell-imprinted poly(dimethylsiloxane) casting procedure could imitate the surface morphology of the plasma membrane, ranging from the nanoscale to the macroscale, which may provide the required topographical cell fingerprints to induce differentiation. Gene expression levels of the genes analyzed (involucrin, collagen type I, and keratin 10) together with protein expression data showed that human adipose-derived stem cells (ADSCs) seeded on these cell-imprinted substrates were driven to adopt the specific shape and characteristics of keratinocytes. The observed morphology of the ADSCs grown on the keratinocyte casts was noticeably different from that of stem cells cultivated on the stem-cell-imprinted substrates. Since the shape and geometry of the nucleus could potentially alter the gene expression, we used molecular dynamics to probe the effect of the confining geometry on the chain arrangement of simulated chromatin fibers in the nuclei. The results obtained suggested that induction of mature cell shapes onto stem cells can influence nucleus deformation of the stem cells followed by regulation of target genes. This might pave the way for a reliable, efficient, and cheap approach of controlling stem cell differentiation toward skin cells for wound healing applications.