Recently, effort has been placed into fabricating model free-floating asymmetric lipid membranes, such as asymmetric vesicles. Here, we report on the use of lipid-coated silica nanoparticles to exchange lipids with initially symmetric vesicles to generate composition-controlled asymmetric vesicles. Our method relies on the simple and natural exchange of lipids between membranes through an aqueous medium. Using a selected temperature, time, and ratio of lipid-coated silica nanoparticles to vesicles, we produced a desired highly asymmetric leaflet composition. At this point, the silica nanoparticles were removed by centrifugation, leaving the asymmetric vesicles in solution. In the present work, the asymmetric vesicles were composed of isotopically distinct dipalmitoylphosphatidylcholine lipids. Lipid asymmetry was detected by both small-angle neutron scattering (SANS) and proton nuclear magnetic resonance ( 1 H NMR). The rate at which the membrane homogenizes at 75 °C was also assessed.

Cholesterol, an essential component in biological membranes, is highly unevenly distributed within the cell, with most localized in the plasma membrane while only a small fraction is found in the endoplasmic reticulum, where it is synthesized. Cellular membranes differ in lipid composition and protein content, and these differences can exist across their leaflets too. This thermodynamic landscape that cellular membranes impose on cholesterol is expected to modulate its transport. To uncover the role the membrane environment has on cholesterol inter- and intra-membrane movement, we used time-resolved small angle neutron scattering to study the passive movement of cholesterol between and within membranes with varying degrees of saturation content. We found that cholesterol moves systematically slower as the degree of saturation in the membranes increases, from a palmitoyl oleyl phosphotidylcholine membrane, which is unsaturated, to a dipalmitoylphosphatidylcholine (DPPC) membrane, which is fully saturated. Additionally, we found that the energetic barrier to move cholesterol in these phosphatidylcholine membranes is independent of their relative lipid composition and remains constant for both flip-flop and exchange at ∼100 kJ/mol. Further, by replacing DPPC with the saturated lipid palmitoylsphingomyelin, an abundant saturated lipid of the outer leaflet of the plasma membrane, we found the rates decreased by a factor of two. This finding is in stark contrast with recent molecular dynamic simulations that predict a dramatic slow-down of seven orders of magnitude for cholesterol flipping in membranes with a similar phosphocholine and SM lipid composition.

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Compartmentalization is a fundamental feature of contemporary biological cells, allowing segregation of the internal content to allow a multitude of incompatible biochemical and biological processes to occur simultaneously. The early formation of compartments within the protocell, the hypothetical prebiotic cell-like precursor, could have created an evolutionary advantage, possibly accelerating the emergence of the first cell. We report here on spontaneous compartmentalization of a solid-surface-adhered phospholipid based protocell model. When a giant phospholipid vesicle, composed of zwitterionic and negatively charged lipids in the presence of multivalent cations, was deposited on silica or aluminum based surfaces, the vesicle membrane wetted the surface as a single intact compartment. Subsequent addition of BAPTA/ EDTA as metal chelator compound, created a chemical gradient, which induced partial de-wetting of the surface and spontaneous compartmentalization of the basal membrane. The transformation resulted in several tens of isolated, intra-vesicular membrane compartments. The observed phenomenon appears to be entirely driven by the membrane-surface interactions. Growth of the compartments and their eventual merging can be accelerated by a mild temperature increase of a few degrees, which is known to weaken the membrane adhesion to the oxide surface. During the formation process, the compartmentalized protocell models can take up and encapsulate watersoluble molecules present in the external environment, where a different internalization rate can be observed for each individual compartment. We show that these processes are entirely determined by fundamental materials properties and interfacial events, and do not require any biological machinery or chemical energy supply. It is therefore conceivable that similar events could have taken place under early Earth conditions, and aided or facilitated the development of primitive life.

Fluorescence correlation spectroscopy (FCS) is one of the prominent tools to elucidate these dynamics in living cells but can only report on the dynamics at one given spatial position at a time. Using scanning fluorescence correlation spectroscopy (sFCS), we obtain a multitude of FCS measurements at different spatial locations. Here, we present a statistical analysis pipeline for sFCS data which allows for the accurate determination of the diffusion dynamics and the differentiation of free (Brownian) from hindered (non-Brownian) diffusion modes. We show free diffusion for phospholipids in model membranes and cells but reveal hindered diffusion of sphingolipids and GPI-anchored proteins in cells. Notably, these measurements can be performed using standard fluorescent dyes or proteins on a conventional confocal laser scanning microscope. To further investigate the dynamics on single cell level, we combine sFCS with stimulated emission depletion (STED) microscopy and by alternating conventional and super-resolved excitation we introduce line interleaved excitation scanning STED-FCS (LIESS-FCS). With LIESS-FCS the diffusion modes can be directly determined at multiple spots within the cellular plasma membrane providing detailed insights into organisation and function. Overall we are presenting a novel toolkit to investigate nano-scale molecular diffusion dynamics for shedding a new light on membrane organisation and heterogeneity. Ordered lipid domains (OLD) are thought to serve as signaling platforms. Here we show that their assembly does not involve intermediates in terms of nonregistered domains in the individual leaflets. The addition of photoswitchable lipids (ceramide PhoDAG-1) to the membrane forming lipid mixture allowed solubilizing and re-assembling OLDs by illuminating freestanding planar lipid bilayers at 365 nm and 475 nm, respectively. Differently colored fluorescent labels adopted a matching distribution in the two monolayers indicating that OLDs from the two leaflets were always in register. Tracing domain diffusion revealed perfect agreement with the Saffman-Delbr€ uck equation. The registration of domains as small as 10 nm in diameter is in line with the predicted role of line tension as a driving force for alignment (1). Registration of the observed micrometer sized domains is mainly driven by membrane undulations (2). 1. Galimzyanov, et al. Phys. Rev. Lett. 2015, 115:088101. 2. Galimzyanov, et al. Biophys. J. 2017. 1617-Plat Asymmetric Membranes and the Study of Lipid Movement across SingleThe great variety of lipid molecules in the cell membrane suggests their complex andunique role in cell function. The cell has further established unique lipid composition indifferent membranes within the cell for directed functionality. In addition, in membranes likethe plasma membrane (PM), there is an asymmetric distribution of lipids between the outer orexoplasmic and the inner or cytoplasmic leaflets and the physiological fate of cells depends onthe strict maintenance of this asymmetry. However, the exact ...