Most eukaryotic cells maintain a large disparity in lipid compositions between the cytosolic and external leaflets of the plasma membrane (PM) bilayer. This lipid asymmetry is maintained by energy-consuming flippase enzymes that selectively drive phospholipids into the cytosolic leaflet, often against large concentration gradients. Scramblases, activated by intracellular Ca2+ or apoptotic signaling, shuttle phospholipids down their concentration gradient to release lipid asymmetry. Such scrambling is typically evidenced by exposure of phosphatidylserine (PS) to the external leaflet and is associated with many physiological processes, most notably blood clotting and cell death, but also activation of immune cells. Here, we show that both PS and phosphatidylethanolamine (PE) appear on the PM external leaflet following immune receptor-mediated activation of mast cells. We also observe similar effects in T cells. Importantly, in contrast to wholesale release of PM asymmetry induced by calcium ionophores or apoptosis, we show that scrambling in activated immune cells is focal, with small, stable regions of surface exposed PS. These scrambled foci are calcium dependent, have lower lipid packing than their surrounding outer leaflet, and are reversible. These observations of local, transient scrambling during physiological activation of healthy immune cells suggest important roles for the lateral and transbilayer organization of membrane lipids.

Membrane lipid oxidation is a universal process that occurs in situations of oxidative stress and is encountered in numerous physiological and pathological situations. Oxidized truncated phospholipids make up a large part of the oxidation products and alter the membrane properties in a way that can lead to cell death. However, the underlying mechanisms are not well understood nor is it clear whether environmental factors, such as pH, can modulate these effects. Using model membranes, we investigate how individual lipid aldehydes and carboxylic acids with truncated acyl chains alter the membrane structure. Our data shows that lipid aldehydes and carboxylic acids have different permeabilization efficiencies towards molecules of varying charge and size and that ΔC9 truncated lipids are usually more efficient in permeabilizing membranes than ΔC5. In terms of physical mechanisms, the ΔC9 truncated lipid carboxylic acid induces permeabilization and membrane curvature in a pH-dependent fashion. This is explained by ionization-dependent exposure of the carboxyl group to the water-bilayer interface, which increases the intrinsic molecular curvature of the oxidized lipid. Conversely, ΔC9 truncated lipid aldehydes and nonionized carboxyls do not induce curved structures but are more efficient in increasing permeability toward larger molecules. We further show that truncated lipids can escape the bilayer and accumulate at interfaces, implying that they might act on neighboring cells. This study indicates that oxidized phospholipids with truncated acyl chains disrupt membrane structure, depending on their specific molecular structure and the pH of the environment, opening a possible route for the design of lipid nanoparticles with pH-dependent drug release.

Plasma membranes are asymmetric, with each monolayer presenting specific lipid compositions and biophysical properties. Transmembrane domains (TMDs) of single-pass transmembrane proteins (spTMPs) have adapted their physico-chemical properties to these asymmetric constraints. In this study, we analysed the structural features of such TMDs across the tree of life to obtain information about their interaction with asymmetric membrane bilayers and predict species-specific membrane properties. We observed that TMDs in the plasma membranes of all eukaryotic species possess asymmetries in lipid accessible surface area (ASA), hydrophobicity, aromaticity and charge. Bacteria deviate from this trend, with strong differences between bacterial clades. Notably, TMDs in the Golgi and the endoplasmic reticulum of eukaryotic species display inverted profiles for accessible surface area, hydrophobicity and aromaticity compared to their plasma-membrane counterparts. To determine how well TMD profiles reflect average membrane properties, we performed molecular dynamics simulations of a spTMP in an asymmetric lipid bilayer whose composition approximates the human plasma membrane. The simulated spTMP was chosen to represent the average TMD properties of the human proteome. We compared the electron density profiles of the simulated asymmetric membrane to the average TMD profiles derived from the human proteome and observed that phospholipid acyl-chain density overlapped very well with TMD hydrophobicity, and phosphate group density with TMD charge. The profiles of phospholipid unsaturation in the acyl chains overlapped well with the average location of TMD phenylalanines in the cytoplasmic leaflet, while there was additional accumulation of large hydrophobic and aromatic residues in the membrane midplane, which had low acyl-chain density. This study reveals the complementarity of membrane and TMD properties in asymmetric membranes, suggesting that the properties of TMDs can be used to make predictions about the properties of their solvating membranes.

Membrane fluidity is a crucial parameter for cellular physiology. Recent evidence suggests that fluidity varies between cell types and states and in diseases. As membrane fluidity has gradually become an important consideration in cell biology and biomedicine, it is essential to have reliable and quantitative ways to measure it in cells. In the past decade, there has been substantial progress both in chemical probes and in imaging tools to make membrane fluidity measurements easier and more reliable. We have recently established a robust pipeline, using confocal imaging and new environment-sensitive probes, that has been successfully used for several studies. Here we present our detailed protocol for membrane fluidity measurement, from labeling to imaging and image analysis. The protocol takes ~4 h and requires basic expertise in cell culture, wet lab and microscopy. Membrane fluidity is a crucial feature in understanding cellular physiology. This protocol describes a robust pipeline for measuring plasma membrane fluidity using confocal imaging and new environment-sensitive probes.

Gram-negative bacteria possess an asymmetric outer membrane (OM) primarily composed of lipopolysaccharides (LPS) on the outer leaflet and phospholipids on the inner leaflet. The outer membrane functions as an effective permeability barrier to compounds such as antibiotics. Studying LPS biosynthesis is therefore helpful to explore novel strategies for new antibiotic development. Metabolic glycan labeling of the bacterial surface has emerged as a powerful method to investigate LPS biosynthesis. However, the previously reported methods of labeling LPS are based on radioactivity or difficult-to-produce analogs of bacterial sugars. In this study, we report on the incorporation of azido galactose into the LPS of the Gram-negative bacteria Escherichia coli and Salmonella typhi via metabolic labeling. As a common sugar analog, azido galactose successfully labeled both O-antigen and core of Salmonella LPS, but not E. coli LPS. This labeling of Salmonella LPS, as shown by SDS-PAGE analysis and fluorescence microscopy, differs from the previously reported labeling of either O-antigen or core of LPS. Our findings are useful for studying LPS biogenesis pathways in Gram-negative bacteria like Salmonella. In addition, our approach is helpful for screening for agents that target LPS biosynthesis as it allows for the detection of newly synthesized LPS that appears in the OM. Furthermore, this approach may also aid in isolating chemically modified LPS for vaccine development or immunotherapy.

Antimicrobial resistance is a leading cause of mortality, calling for the development of new antibiotics. The fungal antibiotic plectasin is a eukaryotic host defence peptide that blocks bacterial cell wall synthesis. Here, using a combination of solid-state nuclear magnetic resonance, atomic force microscopy and activity assays, we show that plectasin uses a calcium-sensitive supramolecular killing mechanism. Efficient and selective binding of the target lipid II, a cell wall precursor with an irreplaceable pyrophosphate, is achieved by the oligomerization of plectasin into dense supra-structures that only form on bacterial membranes that comprise lipid II. Oligomerization and target binding of plectasin are interdependent and are enhanced by the coordination of calcium ions to plectasin's prominent anionic patch, causing allosteric changes that markedly improve the activity of the antibiotic. Structural knowledge of how host defence peptides impair cell wall synthesis will likely enable the development of superior drug candidates. Plectasin, a natural host defence peptide of fungal origin, uses a calcium-sensitive supramolecular killing mechanism to block bacterial cell wall synthesis.

Lipid oxidation is a universal process encountered in many pathophysiological situations. Several oxidized lipids are signalling molecules while others alter cell membrane properties which leads to a loss of membrane function. However, it is not known how a complex mixture of oxidized lipids would alter membrane properties, and how the initial presence of unsaturated lipids would influence this process. We further do not know how certain oxidized lipids can leave the membrane and become signalling molecules.

Oxidative stress in combination with acid stress has been shown to inactivate a wide spectrum of microorganisms, including multi-resistant bacteria. This occurs e.g. in phagolysosomes or during treatment by cold atmospheric pressure plasmas (CAP) and possibly depends on the cell membrane. We therefore explored the effects of CAP-generated reactive oxygen and nitrogen species (RONS) on bacterial growth inhibition and membranes in neutral and acidic suspensions. We observed that growth inhibition was most efficient when bacteria were treated by a mix of short and long-lived RONS in an acidic environment. Membrane packing was affected mainly upon contact with short-lived RONS, while also acidity strongly modulated packing. Under these conditions, Gram-negative bacteria displayed large potassium release while SYTOX Green influx remained marginal. Growth inhibition of Gram-negative bacteria correlated well with outer membrane (OM) permeabilization that occurred upon contact with short and/or long-lived RONS in synergy with acidity. In Gram-positive bacteria, CAP impaired membrane potential possibly through pore formation upon contact with short-lived RONS while formation of membrane protein hydroperoxides was probably involved in these effects. In summary, our study provides a wide perspective on understanding inactivation mechanisms of bacteria by RONS in combination with acidity.

A specific plasma membrane distribution of the mechanosensitive ion channel Piezo1 is required for cell migration, but the mechanism remains elusive. Here, we addressed this question using WT and Piezo1-silenced C2C12 mouse myoblasts and WT and Piezo1-KO human kidney HEK293T cells. We showed that cell migration in a cell-free area and through a porous membrane decreased upon Piezo1 silencing or deletion, but increased upon Piezo1 activation by Yoda1, whereas migration towards a chemoattractant gradient was reduced by Yoda1. Piezo1 organized into clusters, which were preferentially enriched at the front. This polarization was stimulated by Yoda1, accompanied by Ca2+ polarization, and abrogated by partial cholesterol depletion. Piezo1 clusters partially colocalized with cholesterol- and GM1 ganglioside-enriched domains, the proportion of which was increased by Yoda1. Mechanistically, Piezo1 activation induced a differential mobile fraction of GM1 associated with domains and the bulk membrane. Conversely, cholesterol depletion abrogated the differential mobile fraction of Piezo1 associated with clusters and the bulk membrane. In conclusion, we revealed, for the first time, the differential implication of Piezo1 depending on the migration mode and the interplay between GM1/cholesterol-enriched domains at the front during migration in a cell-free area. These domains could provide the optimal biophysical properties for Piezo1 activity and/or spatial dissociation from the PMCA calcium efflux pump.

Lipid oxidation is a universal degradative process of cell membrane lipids that is induced by oxidative stress and reactive oxygen and nitrogen species (RONS) in multiple pathophysiological situations. It has been shown that certain oxidized lipids alter membrane properties, leading to a loss of membrane function. Alteration of membrane properties is thought to depend on the initial membrane lipid composition, such as the number of acyl chain unsaturations. However, it is unclear how oxidative damage is related to biophysical properties of membranes. We therefore set out to quantify lipid oxidation through various analytical methods and determine key biophysical membrane parameters using model membranes containing lipids with different degrees of lipid unsaturation. As source for RONS, we used cold plasma, which is currently developed as treatment for infections and cancer. Our data revealed complex lipid oxidation that can lead to two main permeabilization mechanisms. The first one appears upon direct contact of membranes with RONS and depends on the formation of truncated oxidized phospholipids. These lipids seem to be partly released from the bilayer, implying that they are likely to interact with other membranes and potentially act as signaling molecules. This mechanism is independent of lipid unsaturation, does not rely on large variations in lipid packing, and is most probably mediated via short-living RONS. The second mechanism takes over after longer incubation periods and probably depends on the continued formation of lipid oxygen adducts such as lipid hydroperoxides or ketones. This mechanism depends on lipid unsaturation and involves large variations in lipid packing. This study indicates that polyunsaturated lipids, which are present in mammalian membranes rather than in bacteria, do not sensitize membranes to instant permeabilization by RONS but could promote long-term damage.

Membrane blebbing plays important roles in cell migration, cytokinesis, apoptosis, as well as extracellular vesicle formation for intercellular communication. During budding of the plasma membrane (PM) to form extracellular vesicles, phosphatidylserine is often translocated from the PM inner leaflet to the outer leaflet, indicative of lipid scrambling that results in loss of native membrane asymmetry. Using micrometer-sized giant plasma membrane vesicles (GPMV) as a readout of PM vesiculation, we found that knocking out (KO) the lipid scramblase TMEM16F reduced lipid scrambling and dramatically inhibited GPMV formation, in line with previous observations. TMEM16F repletion could rescue GPMV formation, indicating that lipid scrambling is necessary for membrane budding. We hypothesized three mechanisms to explain this surprising observation, namely that lipid scrambling: (1) depletes PI(4,5)P2 from the inner leaflet leading to detachment of the PM from the actin cytoskeleton, (2) makes PM bilayers softer and easier to bend, (3) externalizes lipids that stabilize highly curved bud necks. Depletion of PI(4,5)P2 was indeed observed during vesiculation, however this occurred in both wild type and KO cells, implying that detachment of PM from actin cytoskeleton due to PIP2 depletion is not sufficient to make large vesicles. Next, through fluorescence lifetime imaging microscopy of a lipid packing reporter (Di4), we found that lipid scrambling leads to a decrease of PM packing, potentially making the membrane softer for bending and thus facilitating membrane budding. Finally, we observed that lipid scrambling resulted in externalization of phosphatidylethanolamine (PE). This PE was preferentially located at the highly curved vesicle necks, suggesting that the intrinsic curvature of PE stabilizes this highly curved membrane structure. Thus, we conclude that lipid scrambling changes membrane biophysical properties, potentially explaining mechanisms underlying vesicle-budding in physiological contexts including apoptotic cell removal, bone mineralization, fertilization, and blood coagulation.

Antibiotics that use novel mechanisms are needed to combat antimicrobial resistance 1 – 3 . Teixobactin 4 represents a new class of antibiotics with a unique chemical scaffold and lack of detectable resistance. Teixobactin targets lipid II, a precursor of peptidoglycan 5 . Here we unravel the mechanism of teixobactin at the atomic level using a combination of solid-state NMR, microscopy, in vivo assays and molecular dynamics simulations. The unique enduracididine C-terminal headgroup of teixobactin specifically binds to the pyrophosphate-sugar moiety of lipid II, whereas the N terminus coordinates the pyrophosphate of another lipid II molecule. This configuration favours the formation of a β-sheet of teixobactins bound to the target, creating a supramolecular fibrillar structure. Specific binding to the conserved pyrophosphate-sugar moiety accounts for the lack of resistance to teixobactin 4 . The supramolecular structure compromises membrane integrity. Atomic force microscopy and molecular dynamics simulations show that the supramolecular structure displaces phospholipids, thinning the membrane. The long hydrophobic tails of lipid II concentrated within the supramolecular structure apparently contribute to membrane disruption. Teixobactin hijacks lipid II to help destroy the membrane. Known membrane-acting antibiotics also damage human cells, producing undesirable side effects. Teixobactin damages only membranes that contain lipid II, which is absent in eukaryotes, elegantly resolving the toxicity problem. The two-pronged action against cell wall synthesis and cytoplasmic membrane produces a highly effective compound targeting the bacterial cell envelope. Structural knowledge of the mechanism of teixobactin will enable the rational design of improved drug candidates.

N-Acetylglucosamine (GlcNAc) is an essential monosaccharide required in almost all organisms. Fluorescent labeling of the peptidoglycan (PG) on N-acetylglucosamine has been poorly explored. Here, we report on the labeling of the PG with a bioorthogonal handle on the GlcNAc. We developed a facile one-step synthesis of uridine diphosphate N-azidoacetylglucosamine (UDP-GlcNAz) using the glycosyltransferase OleD, followed by in vitro incorporation of GlcNAz into the peptidoglycan precursor Lipid II and fluorescent labeling of the azido group via click chemistry. In a PG synthesis assay, fluorescent GlcNAz-labeled Lipid II was incorporated into peptidoglycan by the DD-transpeptidase activity of bifunctional class A penicillin-binding proteins. We further demonstrate the incorporation of GlcNAz into the PG layer of OleD-expressed bacteria by feeding with 2-chloro-4-nitrophenyl GlcNAz (GlcNAz-CNP). Hence, our labeling method using the heterologous expression of OleD is useful to study PG synthesis and possibly other biological processes involving GlcNAc metabolism in vivo.

Cold atmospheric plasma (CAP) has recently emerged as possible treatment for opportunistic infections and cancer. CAP is composed of gas ions, superheated electrons, electric fields and reactive oxygen and nitrogen species (RONS). It can be directly applied to biological tissue because heat generation is low and has further the advantage that its reactive cocktail can be modulated by changing the composition of the feeding gas. CAP has shown to damage cell membranes with some selectivity towards bacterial membranes but its mechanism remains unclear, preventing further development of the technology.

Cleavage of the mammalian plasma protein C4 into C4b initiates opsonization, lysis, and clearance of microbes and damaged host cells by the classical and lectin pathways of the complement system. Dysregulated activation of C4 and other initial components of the classical pathway may cause or aggravate pathologies, such as systemic lupus erythematosus, Alzheimer disease, and schizophrenia. Modulating the activity of C4b by small-molecule or protein-based inhibitors may represent a promising therapeutic approach for preventing excessive inflammation and damage to host cells and tissue. Here, we present seven nanobodies, derived from llama (Lama glama) immunization, that bind to human C4b (Homo sapiens) with high affinities ranging from 3.2 nM to 14 pM. The activity of the nanobodies varies from no to complete inhibition of the classical pathway. The inhibiting nanobodies affect different steps in complement activation, in line with blocking sites for proconvertase formation, C3 substrate binding to the convertase, and regulator-mediated inactivation of C4b. For four nanobodies, we determined single-particle cryo-electron microscopy structures in complex with C4b at 3.4-4 Å resolution. The structures rationalize the observed functional effects of the nanobodies and define their mode of action during complement activation. Thus, we characterized seven anti-C4b nanobodies with diverse effects on the classical pathway of complement activation that may be explored for imaging, diagnostic, or therapeutic applications.