Seipin transmembrane segments critically function in triglyceride nucleation and lipid droplet budding from the membrane

ABSTRACTLipid droplets (LDs) are organelles formed in the endoplasmic reticulum (ER) to store triacylglycerol (TG) and sterol esters. The ER protein seipin is key for LD biogenesis. Seipin forms a cage-like structure, with each seipin monomer containing a conserved hydrophobic helix (HH) and two transmembrane (TM) segments. How the different parts of seipin function in TG nucleation and LD budding is poorly understood. Here, we utilized molecular dynamics simulations of human seipin, along with cell-based experiments, to study seipin’s functions in protein-lipid interactions, lipid diffusion, and LD maturation. All-atom (AA) simulations indicate that most seipin TM segment residues located in the phospholipid (PL) tail region of the bilayer attract TG. We also find seipin TM segments control lipid diffusion and permeation into the protein complex. Simulating larger, growing LDs with coarse-grained (CG) models, we find that the seipin TM segments form a constricted neck structure to facilitate conversion of a flat oil lens into a budding LD. Using cell experiments and simulations, we also show that conserved, positively charged residues at the end of seipin’s TM segments affect LD maturation. We propose a model in which seipin TM segments critically function in TG nucleation and LD growth.

Membrane Constriction by ESCRT-III Proteins Induces Structural Lipid Bilayer Asymmetries

Cells utilize molecular machines to form and remodel their membrane-defined compartments' compositions, shapes, and connections. The regulated activity of these membrane remodeling machines drives processes like vesicular traffic and organelle homeostasis. Although molecular patterning within membranes is essential to cellular life, characterizing the composition and structure of realistic biological membranes on the molecular length scale remains a challenge, particularly during membrane shape transformations. Here, we employed an ESCRT-III protein coating model system to investigate how membrane-binding proteins bind to and alter the structural patterns within lipid bilayers. We observe leaflet-level and localized lipid structures within a constricted and thinned membrane nanotube. To map the fine structure of these membranes, we compared simulated bilayer nanotubes with experimental cryo-EM reconstructions of native membranes and membranes containing halogenated lipid analogs. Halogenated lipids scatter electrons more strongly, and analysis of their surplus scattering enabled us to estimate the concentrations of lipids within each leaflet and to estimate lipid shape and sorting changes induced by high curvature and lipid-protein interactions. Specifically, we found that cholesterol enriched within the inner leaflet due to its spontaneous curvature, while acidic lipids enriched in the outer leaflet due to electrostatic interactions with the protein coat. The docosahexaenoyl (DHA) polyunsaturated chain-containing lipid SDPC enriched strongly at membrane-protein contact sites. Simulations and imaging of brominated SDPC showed how a pair of phenylalanine residues opens a hydrophobic defect in the outer leaflet and how DHA tails stabilize the defect and "snorkel" up to the membrane surface to interact with these side chains. This highly curved nanotube differs markedly from protein-free, flat bilayers in leaflet thickness, lipid diffusion, and other structural asymmetries with implications for our understanding of membrane mechanics.

Surfactant-free production of biomimetic giant unilamellar vesicles using PDMS-based microfluidics

Microfluidic production of giant lipid vesicles presents a paradigm-shift in the development of artificial cells. While production is high-throughput and the lipid vesicles are mono-disperse compared to bulk methods, current technologies rely heavily on the addition of additives such as surfactants, glycerol and even ethanol. Here we present a microfluidic method for producing biomimetic surfactant-free and additive-free giant unilamellar vesicles. The versatile design allows for the production of vesicle sizes ranging anywhere from ~10 to 130 µm with either neutral or charged lipids, and in physiological buffer conditions. Purity, functionality, and stability of the membranes are validated by lipid diffusion, protein incorporation, and leakage assays. Usability as artificial cells is demonstrated by increasing their complexity, i.e., by encapsulating plasmids, smaller liposomes, mammalian cells, and microspheres. This robust method capable of creating truly biomimetic artificial cells in high-throughput will prove valuable for bottom-up synthetic biology and the understanding of membrane function.

Hydration layer of only few molecules controls lipid mobility in biomimetic membranes

Self-assembly of biomembranes results from the intricate interactions between water and the lipids' hydrophilic head groups. Therefore, the lipid-water interplay strongly contributes to modulating membranes architecture, lipid diffusion, and chemical activity. Here, we introduce a new method of obtaining dehydrated, phase-separated, supported lipid bilayers (SLBs) solely by controlling the decrease of their environment's relative humidity. This facilitates the study of the structure and dynamics of SLBs over a wide range of hydration states. We show that the lipid domain structure of phase-separated SLBs is largely insensitive to the presence of the hydration layer. In stark contrast, lipid mobility is drastically affected by dehydration, showing a 6-fold decrease in lateral diffusion. At the same time, the diffusion activation energy increases approximately twofold for the dehydrated membrane. The obtained results, correlated with the hydration structure of a lipid molecule, revealed that about 6-7 water molecules directly hydrating the phosphocholine moiety play a pivotal role in modulating lipid diffusion. These findings could provide deeper insights into the fundamental reactions where local dehydration occurs, for instance during cell-cell fusion, and help us better understand the survivability of anhydrobiotic organisms. Finally, the strong dependence of lipid mobility on the number of hydrating water molecules opens up an application potential for SLBs as very precise, nanoscale hydration sensors.

Interaction of silicone hydrogel contact lenses with lipids – a chronological review

Silicone hydrogel (Si-Hy) contact lenses are a common form of correction of refractive errors and are prescribed by eye care professionals around the world. Si-Hy lenses perform in a complex environment, which is the surface of the eye and the tear film. Therefore, they are exposed to various factors, such as lipid deposits. The aims of this paper are to review available scientific reports on the study of Si-Hy lens interactions with lipids and search for further research objectives. A total of 57 publications were identified and reviewed, from 2003 to 2020. In general, Si-Hy lenses are more likely to accumulate lipid deposits than traditional hydrogel lenses, although there are significant differences between Si-Hy lens materials that may result from different methods used in the studies. The review includes studies on various aspects of interactions between lenses and lipids, such as those concerning the effectiveness of lipids removal from lenses by care solutions. The conclusion points out future research directions, such as measurements of lipid diffusion in Si-Hy lens' matrices.

Bacterial pore-forming proteins induce non-monotonic dynamics due to lipid ejection and crowding

ABSTRACTDeveloping alternate strategies against pore forming toxin (PFT) mediated bacterial virulence factors require an understanding of the target cellular response to combat rising antimicrobial resistance. Membrane-bound protein complexes involving PFTs, released by virulent bacteria are known to form pores leading to host cell lysis. However, membrane disruption and related lipid mediated active repair processes during attack by PFTs remain largely unexplored. We report counter intuitive and non-monotonic variations in lipid diffusion, measured using confocal fluorescence correlation spectroscopy, due to interplay of lipid ejection and crowding by membrane bound oligomers of a prototypical cholesterol dependent cytolysin, Listeriolysin O (LLO). The observed protein concentration dependent dynamical cross-over is correlated with transitions of LLO oligomeric state populations from rings to arc-like pore complexes, predicted using a proposed two-state free area based diffusion model. At low PFT concentrations, a hitherto unexplored regime of increased lipid diffusivity is attributed to lipid ejection events due to a preponderance of ring-like pore states. At higher protein concentrations where membrane inserted arc-like pores dominate, lipid ejection is less efficient and the ensuing crowding results in a lowering of lipid diffusion. These variations in lipid dynamics are corroborated by macroscopic rheological response measurements of PFT bound vesicles. Our study correlates PFT oligomeric state transitions, membrane remodelling and mechanical property variations, providing unique insights into developing strategies to combat virulent bacterial pathogens responsible for several infectious diseases.SIGNIFICANCEDeveloping alternate strategies against pore forming toxin (PFT) mediated bacterial virulence factors requires understanding target cellular responses and cellular defence strategies to combat rising antimicrobial resistant strains. While it is well understood that PFTs exist in a wide variety of oligomeric states, the underlying membrane response to these states is unexplored. Using confocal fluorescence correlation spectroscopy and a membrane free area based model we relate non-monotonic variations in the lipid diffusivity arising from an interplay of lipid ejection events and membrane crowding due to variations in concentration of membrane bound listeriolysin O. Our observations have a direct bearing on understanding cellular defense and repair mechanisms effective during initial stages of bacterial infection and intrinsically connected to the underlying membrane fluidity.

Revealing Plasma Membrane Nano-Domains with Diffusion Analysis Methods

Nano-domains are sub-light-diffraction-sized heterogeneous areas in the plasma membrane of cells, which are involved in cell signalling and membrane trafficking. Throughout the last thirty years, these nano-domains have been researched extensively and have been the subject of multiple theories and models: the lipid raft theory, the fence model, and the protein oligomerization theory. Strong evidence exists for all of these, and consequently they were combined into a hierarchal model. Measurements of protein and lipid diffusion coefficients and patterns have been instrumental in plasma membrane research and by extension in nano-domain research. This has led to the development of multiple methodologies that can measure diffusion and confinement parameters including single particle tracking, fluorescence correlation spectroscopy, image correlation spectroscopy and fluorescence recovery after photobleaching. Here we review the performance and strengths of these methods in the context of their use in identification and characterization of plasma membrane nano-domains.

Surfactant-free production of biomimetic artificial cells using PDMS-based microfluidics

Microfluidic-based production of cellular mimics (e.g. giant vesicles) presents a paradigm-shift in the development of artificial cells. While encapsulation rates are high and vesicles are mono-disperse compared to swelling-based techniques, current microfluidic emulsion-based methods heavily rely on the addition of additives such as surfactants, glycerol and even ethanol to produce stable vesicles. In this work, we present a microfluidic platform designed for the production of cellular mimics in the form of giant unilamellar vesicles (GUVs). Our PDMS-based device comprises a double cross-junction and a serpentine-shaped shear inducing module to produce surfactant-free and additive-free monodisperse biomimetic GUVs. Vesicles can be made with neutral and charged lipids in physiological buffers and, unlike previous works, it is possible to produce them with pure water both inside and outside. By not employing surfactants such as block co-polymers, additives like glycerol, and long-chain poly-vinyl alcohol that are known to alter the properties of lipid membranes, the vesicles are rendered truly biomimetic. The membrane functionality and stability are validated by lipid diffusion, membrane protein incorporation, and leakage assays. To demonstrate the usability of the GUVs using this method, various macromolecules such as DNA, smaller liposomes, mammalian cells and even microspheres are encapsulated within the GUVs.

Nanoscale membrane curvature sorts lipid phases and alters lipid diffusion

ABSTRACTCellular homeostasis requires the precise spatial and temporal control of membrane shape and composition. Membrane regions of high curvature, such as endocytic pits and viral buds, contain distinct lipids and proteins. However, the interplay between membrane curvature and local membrane composition is poorly understood at the nanoscale. Here, we employed single-molecule localization microscopy to observe single-lipid diffusion in model bilayers with varying lipid compositions, phase, temperature, and membrane curvature. Engineered membrane buds were observed for the creation of lateral compositional heterogeneity in otherwise homogeneous membranes. Membrane curvature recruited liquid-disordered lipid phases in phase-separated membranes and altered the diffusion of the lipids. Supported lipid bilayers were created over 50-nm radius nanoparticles to engineer nanoscale membrane curvature that mimics the size of naturally occurring endocytic pits and viral buds. The disorder-preferring lipids sorted to the nanoscale curvature at all temperatures, but only when embedded in a membrane capable of sustaining liquid-liquid phase separation at low temperatures. This result suggests that lipid sorting by the membrane curvature was only possible when coupled with lipid phase separation. The curvature affected the local membrane composition most strongly when the curvature was locally surrounded by a liquid-ordered phase typically associated with a stiffer bending modulus. The curvature-induced sorting of lipid phases was quantified by the sorting of disorder-preferring fluorescent lipids, single-lipid diffusion measurements, and simulations that couple the lipid phase separation to the membrane shape. Unlike single-component membranes, lipids in phase-separated membranes demonstrated faster diffusion on curved membranes than the surrounding, flat membrane. These results demonstrate that curvature-induced membrane compositional heterogeneity can be achieved by collective behavior with lipid phase separation when single-molecule properties (i.e., packing parameter) are insufficient. These results support the hypothesis that the coupling of lipid phases and membrane shape may yield lateral membrane composition heterogeneities with functional consequences.STATEMENT OF SIGNIFICANCENanoscopic membrane organization and dynamics are critical for cellular function but challenging to experimentally measure. This work brings together super-resolution optical methods with engineered substrates to reveal the interplay between curvature, composition, phase, and diffusion in model membranes. We report that curvature can induce phase separation in otherwise homogeneous membranes and that the phase-curvature coupling has a direct implication on lipid mobility. In sum, this discovery advances our understanding of the fundamental membrane biophysics that regulate membrane activities such as endocytosis and viral budding.