Molecular probes for tracking lipid droplet membrane dynamics.

Lipid droplets (LDs) feature a unique monolayer lipid membrane that has not been extensively studied due to the lack of suitable molecular probes that are able to distinguish this membrane from the LD lipid core. In this work, we present a three-pronged molecular probe design strategy that combines lipophilicity-based organelle targeting with microenvironment-dependent activation and design an LD membrane labeling pro-probe called LDM. Upon activation by the HClO/ClO- microenvironment that surrounds LDs, LDM pro-probe releases LDM-OH probe that binds to LD membrane proteins thus enabling visualization of the ring-like LD membrane. By utilizing LDM, we identify the dynamic mechanism of LD membrane contacts and their protein accumulation parameters. Taken together, LDM represents the first molecular probe for imaging LD membranes in live cells to the best of our knowledge, and represents an attractive tool for further investigations into the specific regulatory mechanisms with LD-related metabolism diseases and drug screening.

Coarse-Grained Simulations of Adeno-Associated Virus and Its Receptor Reveal Influences on Membrane Lipid Organization and Curvature.

Adeno-associated virus (AAV) is a well-known gene delivery tool with a wide range of applications, including as a vector for gene therapies. However, the molecular mechanism of its cell entry remains unknown. Here, we performed coarse-grained molecular dynamics simulations of the AAV serotype 2 (AAV2) capsid and the universal AAV receptor (AAVR) in a model plasma membrane environment. Our simulations show that binding of the AAV2 capsid to the membrane induces membrane curvature, along with the recruitment and clustering of GM3 lipids around the AAV2 capsid. We also found that the AAVR binds to the AAV2 capsid at the VR-I loops using its PKD2 and PKD3 domains, whose binding poses differs from previous structural studies. These first molecular-level insights into AAV2 membrane interactions suggest a complex process during the initial phase of AAV2 capsid internalization.

Cadmium and calcium ions' effects on the growth of Pleurotus ostreatus mycelia are related to phosphatidylethanolamine content.

Heavy metal Cd2+ can easily be accumulated by fungi, causing significant stress, with the fungal cell membrane being one of the primary targets. However, the understanding of the mechanisms behind this stress remains limited. This study investigated the changes in membrane lipid molecules of Pleurotus ostreatus mycelia under Cd2+ stress and the antagonistic effect of Ca2+ on this stress. Cd2+ in the growth media significantly inhibited mycelial growth, with increasing intensity at higher concentrations. The addition of Ca2+ mitigated this Cd2+-induced growth inhibition. Lipidomic analysis showed that Cd2+ reduced membrane lipid content and altered lipid composition, while Ca2+ counteracted these changes. The effects of both Cd2+ and Ca2+ on lipids are dose dependent and phosphatidylethanolamine appeared most affected. Cd2+ also caused a phosphatidylcholine/phosphatidylethanolamine ratio increase at high concentrations, but Ca2+ helped maintain normal levels. The acyl chain length and unsaturation of lipids remained unaffected, suggesting Cd2+ doesn't alter acyl chain structure of lipids. These findings suggest that Cd2+ may affect the growth of mycelia by inhibiting the synthesis of membrane lipids, particular the synthesis of phosphatidylethanolamine, providing novel insights into the mechanisms of Cd2+ stress in fungi and the role of Ca2+ in mitigating the stress.

Making lipids very unhappy to discover how they bind to proteins.

Membrane lipid composition is maintained by conserved lipid transfer proteins, but computational approaches to study their lipid-binding mechanisms are limiting. Srinivasan et al. (https://doi.org/10.1083/jcb.202312055) develop a clever molecular dynamics simulations assay to accurately model lipid-binding poses in lipid transfer proteins.

Substrate engagement by the intramembrane metalloprotease SpoIVFB.

S2P intramembrane metalloproteases regulate diverse signaling pathways across all three domains of life. However, the mechanism by which S2P metalloproteases engage substrates and catalyze peptide hydrolysis within lipid membranes has remained elusive. Here we determine the cryo-EM structure of the S2P family intramembrane metalloprotease SpoIVFB from Bacillus subtilis bound to its native substrate Pro-σK. The structure and accompanying biochemical data demonstrate that SpoIVFB positions Pro-σK at the enzyme active site through a ß-sheet augmentation mechanism, and reveal key interactions between Pro-σK and the interdomain linker connecting SpoIVFB transmembrane and CBS domains. The cryo-EM structure and molecular dynamics simulation reveal a plausible path for water to access the membrane-buried active site of SpoIVFB, and suggest a possible role of membrane lipids in facilitating substrate capture. These results provide key insight into how S2P intramembrane metalloproteases capture and position substrates for hydrolytic proteolysis within the hydrophobic interior of a lipid membrane.

Membrane Activity and Viroporin Assembly for the SARS-CoV-2 E Protein Are Regulated by Cholesterol.

The SARS-CoV-2 E protein is an enigmatic viral structural protein with reported viroporin activity associated with the acute respiratory symptoms of COVID-19, as well as the ability to deform cell membranes for viral budding. Like many viroporins, the E protein is thought to oligomerize with a well-defined stoichiometry. However, attempts to determine the structure of the protein complex have yielded inconclusive results, suggesting several possible oligomers, ranging from dimers to pentamers. Here, we combined patch-clamp, confocal fluorescence microscopy on giant unilamellar vesicles, and atomic force microscopy to show that E protein can exhibit two modes of membrane activity depending on membrane lipid composition. In the absence or the presence of a low content of cholesterol, the protein forms short-living transient pores, which are seen as semi-transmembrane defects in a membrane by atomic force microscopy. Approximately 30 mol% cholesterol is a threshold for the transition to the second mode of conductance, which could be a stable pentameric channel penetrating the entire lipid bilayer. Therefore, the E-protein has at least two different types of activity on membrane permeabilization, which are regulated by the amount of cholesterol in the membrane lipid composition and could be associated with different types of protein oligomers.

FabR, a regulator of membrane lipid homeostasis, is involved in Klebsiella pneumoniae biofilm robustness.

Biofilm is a dynamic structure from which individual bacteria and micro-aggregates are released to subsequently colonize new niches by either detachment or dispersal. Screening of a transposon mutant library identified genes associated with the alteration of Klebsiella pneumoniae biofilm including fabR, which encodes a transcriptional regulator involved in membrane lipid homeostasis. An isogenic ∆fabR mutant formed more biofilm than the wild-type (WT) strain and its trans-complemented strain. The thick and round aggregates observed with ∆fabR were resistant to extensive washes, unlike those of the WT strain. Confocal microscopy and BioFlux microfluidic observations showed that fabR deletion was associated with biofilm robustness and impaired erosion over time. The genes fabB and yqfA associated with fatty acid metabolism were significantly overexpressed in the ∆fabR strain, in both planktonic and biofilm conditions. Two monounsaturated fatty acids, palmitoleic acid (C16:1) and oleic acid (C18:1), were found in higher proportion in biofilm cells than in planktonic forms, whereas heptadecenoic acid (C17:1) and octadecanoic acid, 11-methoxy (C18:0-OCH3) were found in higher proportion in the planktonic lifestyle. The fabR mutation induced variations in the fatty acid composition, with no clear differences in the amounts of saturated fatty acids (SFA) and unsaturated fatty acids for the planktonic lifestyle but lower SFA in the biofilm form. Atomic force microscopy showed that deletion of fabR is associated with decreased K. pneumoniae cell rigidity in the biofilm lifestyle, as well as a softer, more elastic biofilm with increased cell cohesion compared to the wild-type strain.IMPORTANCEKlebsiella pneumoniae is an opportunistic pathogen responsible for a wide range of nosocomial infections. The success of this pathogen is due to its high resistance to antibiotics and its ability to form biofilms. The molecular mechanisms involved in biofilm formation have been largely described but the dispersal process that releases individual and aggregate cells from mature biofilm is less well documented while it is associated with the colonization of new environments and thus new threats. Using a multidisciplinary approach, we show that modifications of bacterial membrane fatty acid composition lead to variations in the biofilm robustness, and subsequent bacterial detachment and biofilm erosion over time. These results enhance our understanding of the genetic requirements for biofilm formation in K. pneumoniae that affect the time course of biofilm development and the embrittlement step preceding its dispersal that will make it possible to control K. pneumoniae infections.

Lipid redistribution due to a cell-cell fusion pore.

We consider the redistribution of lipids comprising the plasma membranes during cell-cell fusion, particularly due to the presence of a fusion pore. Assuming the membranes are of constant thickness, we find that the mole fraction of cholesterol increases in the directly apposed exoplasmic leaflets, and is decreased in the cytoplasmic leaflets. The redistribution of the phospholipids is obtained as well.

Cholesterol-Dependent Serotonin Insertion Controlled by Gangliosides in Model Lipid Membranes.

Serotonin is distinct among synaptic neurotransmitters because it is amphipathic and released from synaptic vesicles at concentrations superior to its water solubility limit (270 mM in synaptic vesicles for a solubility limit of 110 mM). Hence, serotonin is mostly aggregated in the synaptic cleft, due to extensive aromatic stacking. This important characteristic has received scant attention, as most representations of the serotonergic synapse take as warranted that serotonin molecules are present as monomers after synaptic vesicle exocytosis. Using a combination of in silico and physicochemical approaches and a new experimental device mimicking synaptic conditions, we show that serotonin aggregates are efficiently dissolved by gangliosides (especially GM1) present in postsynaptic membranes. This initial interaction, driven by electrostatic forces, attracts serotonin from insoluble aggregates and resolves micelles into monomers. Serotonin also interacts with cholesterol via a set of CH-π and van der Waals interactions. Thus, gangliosides and cholesterol act together as a functional serotonin-collecting funnel on brain cell membranes. Based on this unique mode of interaction with postsynaptic membranes, we propose a new model of serotonergic transmission that takes into account the post-exocytosis solubilizing effect of gangliosides and cholesterol on serotonin aggregates.

Lipid lateral diffusion: mechanisms and modulators.

The lateral diffusion of lipids within a membrane is of paramount importance, serving as a central mechanism in numerous physiological processes including cell signaling, membrane trafficking, protein activity regulation, and energy transduction pathways. This review offers a comprehensive overview of lateral lipid diffusion in model biomembrane systems explored through the lens of neutron scattering techniques. We examine diverse models of lateral diffusion and explore the various factors influencing this fundamental process in membrane dynamics. Additionally, we offer a thorough summary of how different membrane-active compounds, including drugs, antioxidants, stimulants, and membrane proteins, affect lipid lateral diffusion. Our analysis unveils the intricate interplay between these additives and membranes, shedding light on their dynamic interactions. We elucidate that this interaction is governed by a complex combination of multiple factors including the physical state and charge of the membrane, the concentration of additives, the molecular architecture of the compounds, and their spatial distribution within the membrane. In conclusion, we briefly discuss the future directions and areas requiring further investigation in the realm of lateral lipid diffusion, highlighting the need to study more realistic membrane systems.

Polyunsaturated triacylglycerol accumulation mainly attributes to turnover of de novo-synthesized membrane lipids in stress-induced starchless Chlamydomonas.

KEY MESSAGE: Assembly of PUFA-attached TAGs is intimately correlated to turnover of newly formed membrane lipids in starch-deficient Chlamydomonas exposed to high light and nitrogen stress under air-aerated mixotrophic conditions. Triacylglycerols (TAGs) rich in polyunsaturated fatty acids (PUFAs) in microalgae have attracted extensive attention due to its promising application in nutraceuticals and other high-value compounds. Previous studies revealed that PUFAs accumulated in TAG primarily derived from the dominant membrane lipids, monogalactosyldiacylglycerolipid, digalactosyldiacylglycerol and diacylglycerol-N,N,N-trimethylhomoserine (DGTS), in the model alga Chlamydomonas reinhardtii. However, their respective contribution to PUFA-attached TAG integration has not been clearly deciphered, particularly in starchless Chlamydomonas that hyper-accumulates TAG. In this study, the starchless C. reinhardtii BAFJ5 was mixotrophically cultivated in photobioreactors aerated with air (0.04% CO2), and we monitored the dynamic changes in growth, cellular carbon and nitrogen content, photosynthetic activity, biochemical compositions, and glycerolipid remodeling under high light and nitrogen starvation conditions. The results indicated that multiple PUFAs continually accumulated in total lipids and TAG, and the primary distributors of these PUFAs gradually shifted from membrane lipids to TAG in stress-induced BAFJ5. The stoichiometry analyses showed that the PUFA-attached TAG assembly attributed to turnover of not only the major glycerolipids, but also the phospholipids, phosphatidylethanolamine (PE) and phosphatidylglycerol. Specifically, the augmented C16:3n3 and C18:3n3 in TAG mainly originated from de novo-synthesized galactolipids, while the cumulative C18:3n6 and C18:4n3 in TAG were intimately correlated with conversion of the newly formed DGTS and PE. These findings emphasized significance of PUFA-attached TAG formation dependent on turnover of de novo assembled membrane lipids in starch-deficient Chlamydomonas, beneficial for enhanced production of value-added lipids in microalgae.

HIV-1 Gag Polyprotein Affinity to the Lipid Membrane Is Independent of Its Surface Charge.

The binding of the HIV-1 Gag polyprotein to the plasma membrane is a critical step in viral replication. The association with membranes depends on the lipid composition, but its mechanisms remain unclear. Here, we report the binding of non-myristoylated Gag to lipid membranes of different lipid compositions to dissect the influence of each component. We tested the contribution of phosphatidylserine, PI(4,5)P2, and cholesterol to membrane charge density and Gag affinity to membranes. Taking into account the influence of the membrane surface potential, we quantitatively characterized the adsorption of the protein onto model lipid membranes. The obtained Gag binding constants appeared to be the same regardless of the membrane charge. Furthermore, Gag adsorbed on uncharged membranes, suggesting a contribution of hydrophobic forces to the protein-lipid interaction. Charge-charge interactions resulted in an increase in protein concentration near the membrane surface. Lipid-specific interactions were observed in the presence of cholesterol, resulting in a two-fold increase in binding constants. The combination of cholesterol with PI(4,5)P2 showed cooperative effects on protein adsorption. Thus, we suggest that the affinity of Gag to lipid membranes results from a combination of electrostatic attraction to acidic lipids, providing different protein concentrations near the membrane surface, and specific hydrophobic interactions.

ER-plasma membrane contact sites deliver ER lipids and proteins for rapid cell surface expansion.

As a consequence of hypoosmotic shock, yeast cells swell rapidly and increase the surface area by ∼20% in 20 s. Approximately, 35% of this surface increase is mediated by the ER-plasma membrane contact sites, specifically the tricalbins, which are required for the delivery of both lipids and the GPI-anchored protein Crh2 from the cortical ER to the plasma membrane. Therefore, we propose a new function for the tricalbins: mediating the fusion of the ER to the plasma membrane at contact sites. This proposed fusion is triggered by calcium influx via the stretch-gated channel Cch1 and is supported by the anoctamin Ist2.

Entangling roles of cholesterol-dependent interaction and cholesterol-mediated lipid phase heterogeneity in regulating listeriolysin O pore-formation.

Cholesterol-dependent cytolysins (CDCs) are the distinct class of ß-barrel pore-forming toxins (ß-PFTs) that attack eukaryotic cell membranes, and form large, oligomeric, transmembrane ß-barrel pores. Listeriolysin O (LLO) is a prominent member in the CDC family. As documented for the other CDCs, membrane cholesterol is essential for the pore-forming functionality of LLO. However, it remains obscure how exactly cholesterol facilitates its pore formation. Here, we show that cholesterol promotes both membrane-binding and oligomerization of LLO. We demonstrate cholesterol not only facilitates membrane-binding, it also enhances the saturation threshold of LLO-membrane association, and alteration of the cholesterol-recognition motif in the LLO mutant (LLOT515G-L516G) compromises its pore-forming efficacy. Interestingly, such defect of LLOT515G-L516G could be rescued in the presence of higher membrane cholesterol levels, suggesting cholesterol can augment the pore-forming efficacy of LLO even in the absence of a direct toxin-cholesterol interaction. Furthermore, we find the membrane-binding and pore-forming abilities of LLOT515G-L516G, but not those of LLO, correlate with the cholesterol-dependent rigidity/ordering of the membrane lipid bilayer. Our data further suggest that the line tension derived from the lipid phase heterogeneity of the cholesterol-containing membranes could play a pivotal role in LLO function, particularly in the absence of cholesterol binding. Therefore, in addition to its receptor-like role, we conclude cholesterol can further facilitate the pore-forming, membrane-damaging functionality of LLO by asserting the optimal physicochemical environment in membranes. To the best of our knowledge, this aspect of the cholesterol-mediated regulation of the CDC mode of action has not been appreciated thus far.

Analysis of lipid composition of cell-bound membrane vesicles (CBMVs) derived from endothelial cells.

Cell-bound membrane vesicles (CBMVs), a novel type of membrane vesicles, have been identified through a series of characterization tools. However, the lipid composition of CBMVs has not yet been characterized. This study focuses on the differences in lipid composition between CBMVs and cell membranes. In order to determine the lipid composition of CBMVs and cell membranes of Human umbilical vein endothelial cells (HUVECs) and find out differential metabolites, this study was carried out by isolating CBMVs lipids and characterizing them using high-performance liquid chromatography tandem secondary mass spectrometry (LC-MS/MS). The results showed the presence of 213 up-regulated and 726 down-regulated lipids in CBMVs compared to cell membranes which produced CBMVs. There are lipids expressed in CBMVs and not in cell membranes: DGDG 18:0_8:0, DGDG O-8:0_16:1, DGDG O-26:7_26:7, DGDG O-16:3_26:7, TG 15:4_21:5_22:5; 4O, PC 49:11, PG 19:5_38:10, PI 60:14, PI 44:9, PI 25:2, PI 43:5, PI 50:10, PS 55:10. DGDG (digalactosyl diglyceride), MGDG (monogalactosyl diglyceride) belongs to galactosyl diglyceride, promotes fat catabolism, which also has antioxidant and anti-inflammatory effects, and unsaturated diacylglycerols are a class of antioxidant compounds, which enables CBMVs to have a therapeutic potential.

Curvature-Assisted Vesicle Explosion Under Light-Induced Asymmetric Oxidation.

Exposure of cell membranes to reactive oxygen species can cause oxidation of membrane lipids. Oxidized lipids undergo drastic conformational changes, compromising the mechanical integrity of the membrane and causing cell death. For giant unilamellar vesicles, a classic cell mimetic system, a range of mechanical responses under oxidative assault has been observed including formation of nanopores, transient micron-sized pores, and total sudden catastrophic collapse (i.e., explosion). However, the physical mechanism regarding how lipid oxidation causes vesicles to explode remains elusive. Here, with light-induced asymmetric oxidation experiments, the role of spontaneous curvature on vesicle instability and its link to the conformational changes of oxidized lipid products is systematically investigated. A comprehensive membrane model is proposed for pore-opening dynamics incorporating spontaneous curvature and membrane curling, which captures the experimental observations well. The kinetics of lipid oxidation are further characterized and how light-induced asymmetric oxidation generates spontaneous curvature in a non-monotonic temporal manner is rationalized. Using the framework, a phase diagram with an analytical criterion to predict transient pore formation or catastrophic vesicle collapse is provided. The work can shed light on understanding biomembrane stability under oxidative assault and strategizing release dynamics of vesicle-based drug delivery systems.

Protocol for detecting nitrative stress in biological lipid membranes in murine cells and tissues.

Detection of nitrative stress is crucial to understanding redox signaling and pathophysiology. Dysregulated nitrative stress, which generates high levels of peroxynitrite, can damage lipid membranes and cause activation of proinflammatory pathways associated with pulmonary complications. Here, we present a protocol for implementing a peroxynitrite-sensing phospholipid to investigate nitrative stress in murine cells and lung tissue. We detail procedures for sensing ONOO- in stimulated cells, both ex vivo and in vivo, using murine models of acute lung injury (ALI). For complete details on the use and execution of this protocol, please refer to Gutierrez and Aggarwal et al.1.

Metabolic and gene-expression analyses reveal developmental dynamics of cutin deposition in pomegranate fruit grown under different environmental conditions.

The chemical and transcriptional changes in the cuticle of pomegranate (Punica granatum L.) fruit grown under different environmental conditions were studied. We collected fruit from three orchards located in different regions in Israel, each with a distinct microclimate. Fruit were collected at six phenological stages, and cutin monomers in the fruit cuticle were profiled by gas chromatography-mass spectrometry (GC-MS), along with qPCR transcript-expression analyses of selected cutin-related genes. While fruit phenotypes were comparable along development in all three orchards, principal component analyses of cutin monomer profiles suggested clear separation between cuticle samples of young green fruit to those of maturing fruit. Moreover, total cutin contents in green fruit were lower in the orchard characterized by a hot and dry climate compared to orchards with moderate temperatures. The variances detected in total cutin contents between orchards corresponded well with the expression patterns of BODYGUARD, a key biosynthetic gene operating in the cutin biosynthetic pathway. Based on our extraction protocols, we found that the cutin polyester that builds the pomegranate fruit cuticle accumulates some levels of gallic acid-the precursor of punicalagin, a well-known potent antioxidant metabolite in pomegranate fruit. The gallic acid was also one of the predominant metabolites contributing to the variability between developmental stages and orchards, and its accumulation levels were opposite to the expression patterns of the UGT73AL1 gene which glycosylates gallic acid to synthesize punicalagin. To the best of our knowledge, this is the first detailed composition of the cutin polyester that forms the pomegranate fruit cuticle.

Rhamnolipids and fengycins interact differently with biomimetic lipid membrane models of Botrytis cinerea and Sclerotinia sclerotiorum: Lipidomics profiles and biophysical studies.

Rhamnolipids (RLs) and Fengycins (FGs) are biosurfactants with very promising antifungal properties proposed to reduce the use of synthetic pesticides in crops. They are amphiphilic molecules, both known to target the plasma membrane. They act differently on Botrytis cinerea and Sclerotinia sclerotiorum, two close Sclerotiniaceae phytopathogenic fungi. RLs are more efficient at permeabilizing S. sclerotiorum, and FGs are more efficient at permeabilizing B. cinerea mycelial cells. To study the link between the lipid membrane composition and the activity of RLs and FGs, we analyzed the lipid profiles of B. cinerea and S. sclerotiorum. We determined that unsaturated or saturated C18 and saturated C16 fatty acids are predominant in both fungi. We also showed that phosphatidylethanolamine (PE), phosphatidic acid (PA), and phosphatidylcholine (PC) are the main phospholipids (in this order) in both fungi, with more PA and less PC in S. sclerotiorum. The results were used to build biomimetic lipid membrane models of B. cinerea and S. sclerotiorum for all-atom molecular dynamic simulations and solid-state NMR experiments to more deeply study the interactions between RLs or FGs with different compositions of lipid bilayers. Distinctive effects are exerted by both compounds. RLs completely insert in all the studied model membranes with a fluidification effect. FGs tend to form aggregates out of the bilayer and insert individually more easily into the models representative of B. cinerea than those of S. sclerotiorum, with a higher fluidification effect. These results provide new insights into the lipid composition of closely related fungi and its impact on the mode of action of very promising membranotropic antifungal molecules for agricultural applications.

Optimizing Archaeal Lipid Biosynthesis in Escherichia coli.

Membrane lipid chemistry is remarkably different in archaea compared with bacteria and eukaryotes. In the evolutionary context, this is also termed the lipid divide and is reflected by distinct biosynthetic pathways. Contemporary organisms have almost without exception only one type of membrane lipid. During early membrane evolution, mixed membrane stages likely occurred, and it was hypothesized that the instability of such mixtures was the driving force for the lipid divide. To examine the compatibility between archaeal and bacterial lipids, the bacterium Escherichia coli has been engineered to contain both types of lipids with varying success. Only limited production of archaeal lipid archaetidylethanolamine was achieved. Here, we substantially increased its production in E. coli by overexpression of an archaeal phosphatidylserine synthase needed for ethanolamine headgroup attachment. Furthermore, we introduced a synthetic isoprenoid utilization pathway to increase the supply of isopentenyl-diphosphate and dimethylallyl diphosphate. This improved archaeal lipid production substantially. The archaeal phospholipids also served as a substrate for the E. coli cardiolipin synthase, resulting in archaeal and novel hybrid archaeal/bacterial cardiolipin species not seen in living organisms before. Growth of the E. coli strain with the mixed membrane shows an enhanced sensitivity to the inhibitor of fatty acid biosynthesis, cerulenin, indicating a critical dependence of the engineered E. coli strain on its native phospholipids.