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.

Data of the osmotic water permeability of a lipid bilayer (diphytanoylphosphaticylcholin) in the presence of cholesterol (30 mole%) are shown under the simultaneous measurement of bilayer tension. Detailed methods and procedures for evaluating the water permeability using the moving membrane method (K. Yano, M. Iwamoto, T. Koshiji & S. Oiki: Visualizing the Osmotic Water Permeability of a Lipid Bilayer under Measured Bilayer Tension Using a Moving Membrane Method. Journal of Membrane Science, 627 (2021) 119231) are presented. The planar lipid bilayer is formed in a glass capillary, separating two aqueous compartments with different osmolarities, and osmotically-driven water flux is visualized as membrane movements along the capillary. The water permeability was evaluated under constant membrane area and tension after correcting for the unstirred layer effect. In these measurements, geometrical features, such as the edge of the planar lipid bilayer and the contact angle between bilayer and monolayer, were image-analyzed. The unstirred layer was evaluated electrophysiologically, in which gramicidin A channel was employed. In the presence of an osmotic gradient, the gramicidin channel generates the streaming potential, and the measured streaming potential data and the derived water-ion coupling ratio (water flux/ion flux) are shown. Detailed descriptions of the integrated method of the moving membrane allow researchers to reproduce the experiment and give opportunities to examine water permeability of various types of membranes, including those containing aquaporins. The present data of osmotic water permeability are compared with the previously published data, while they neglected the bilayer tension.

Once membrane potential changes or ligand binding activates the ion channel, the activity of the channel is finely modulated by the fluctuating membrane environment, involving local lipid composition and membrane tension. In the age of post-structural biology, the factors in the membrane that affect the ion channel function and how they affect it are a central concern among ion channel researchers. This review presents our strategies for elucidating the molecular mechanism of membrane effects on ion channel activity. The membrane\'s diverse and intricate effects consist of chemical and physical processes. These elements can be quantified separately using lipid bilayer methods, in which a membrane is reconstructed only from the components of interest. In our advanced lipid bilayer platform (contact bubble bilayer, CBB), physical features of the membrane, such as tension, are freely controlled. We have elucidated how the specific lipid or membrane tension modulates the gating of a prototypical potassium channel, KcsA, embedded in the lipid bilayer. Our results reveal the molecular mechanism of the channel for sensing and responding to the membrane environment.

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.

Molecular mechanisms underlying channel-membrane interplay have been extensively studied. Cholesterol, as a major component of the cell membrane, participates either in specific binding to channels or via modification of membrane physical features. Here, we examined the action of various sterols (cholesterol, epicholesterol, etc.) on a prototypical potassium channel (KcsA). Single-channel current recordings of the KcsA channel were performed in a water-in-oil droplet bilayer (contact bubble bilayer) with a mixed phospholipid composition (azolectin). Upon membrane perfusion of sterols, the activated gate at acidic pH closed immediately, irrespective of the sterol species. During perfusion, we found that the contacting bubbles changed their shapes, indicating alterations in membrane physical features. Absolute bilayer tension was measured according to the principle of surface chemistry, and inherent bilayer tension was ∼5 mN/m. All tested sterols decreased the tension, and the nonspecific sterol action to the channel was likely mediated by the bilayer tension. Purely mechanical manipulation that reduced bilayer tension also closed the gate, whereas the resting channel at neutral pH never activated upon increased tension. Thus, rather than conventional stretch activation, the channel, once ready to activate by acidic pH, changes the open probability through the action of bilayer tension. This constitutes a channel regulating modality by two successive stimuli. In the contact bubble bilayer, inherent bilayer tension was high, and the channel remained boosted. In the cell membrane, resting tension is low, and it is anticipated that the ready-to-activate channel remains closed until bilayer tension reaches a few millinewton/meter during physiological and pathological cellular activities.