Rewiring phospholipid biosynthesis reveals resilience to membrane perturbations and uncovers regulators of lipid homeostasis.

The organelles of eukaryotic cells differ in their membrane lipid composition. This heterogeneity is achieved by the localization of lipid synthesizing and modifying enzymes to specific compartments, as well as by intracellular lipid transport that utilizes vesicular and non-vesicular routes to ferry lipids from their place of synthesis to their destination. For instance, the major and essential phospholipids, phosphatidylethanolamine (PE) and phosphatidylcholine (PC), can be produced by multiple pathways and, in the case of PE, also at multiple locations. However, the molecular components that underlie lipid homeostasis as well as the routes allowing their distribution remain unclear. Here, we present an approach in which we simplify and rewire yeast phospholipid synthesis by redirecting PE and PC synthesis reactions to distinct subcellular locations using chimeric enzymes fused to specific organelle targeting motifs. In rewired conditions, viability is expected to depend on homeostatic adaptation to the ensuing lipostatic perturbations and on efficient interorganelle lipid transport. We therefore performed genetic screens to identify factors involved in both of these processes. Among the candidates identified, we find genes linked to transcriptional regulation of lipid homeostasis, lipid metabolism, and transport. In particular, we identify a requirement for Csf1-an uncharacterized protein harboring a Chorein-N lipid transport motif-for survival under certain rewired conditions as well as lipidomic adaptation to cold, implicating Csf1 in interorganelle lipid transport and homeostatic adaptation.

The stressed life of a lipid in the Zika virus membrane.

Protein-lipid interactions modulate a plethora of physiopathologic processes and have been the subject of countless studies. However, these kinds of interactions in the context of viral envelopes have remained relatively unexplored, partially because the intrinsically small dimensions of the molecular systems escape to the current resolution of experimental techniques. However, coarse-grained and multiscale simulations may fill that niche, providing nearly atomistic resolution at an affordable computational price. Here we use multiscale simulations to characterize the lipid-protein interactions in the envelope of the Zika Virus, a prominent member of the Flavivirus genus. Comparisons between the viral envelope and simpler molecular systems indicate that the viral membrane is under extreme pressures and asymmetric forces. Furthermore, the dense net of protein-protein contacts established by the envelope proteins creates poorly solvated regions that destabilize the external leaflet leading to a decoupled dynamics between both membrane layers. These findings lead to the idea that the Flaviviral membrane may store a significant amount of elastic energy, playing an active role in the membrane fusion process.

The enigma of phosphoinositides and their derivatives: Their role in regulation of subcellular compartment morphology.

The general segregation of a molecular class, lipids, from the pathways of cellular communication, via endo-membranes, has resulted in the over-simplification and misconceptions in deciphering cell signalling mechanisms. Mechanisms in signal transduction and protein activation require targeting of proteins to membranous compartments with a specific localised morphology and dynamics that are dependent on their lipid composition. Many posttranslational events define cellular behaviours and without the active role of membranous compartments these events lead to various dysregulations of the signalling pathways. We summarise the key findings, using tools such as the rapalogue dimerisation, in the structural roles and signalling of the inter-related phosphoinositide lipids and their derivative, diacylglycerol, in the regulation of nuclear envelope biogenesis and other subcellular compartments such as the nucleoplasmic reticulum.

Using cyclodextrin-induced lipid substitution to study membrane lipid and ordered membrane domain (raft) function in cells.

Methods for efficient cyclodextrin-induced lipid exchange have been developed in our lab. These make it possible to almost completely replace the lipids in the outer leaflet of artificial membranes or the plasma membranes of living cells with exogenous lipids. Lipid replacement/substitution allows detailed studies of how lipid composition and asymmetry influence the structure and function of membrane domains and membrane proteins. In this review, we both summarize progress on cyclodextrin exchange in cells, mainly by the use of methyl-alpha cyclodextrin to exchange phospholipids and sphingolipids, and discuss the issues to consider when carrying out lipid exchange experiments upon cells. Issues that impact interpretation of lipid exchange are also discussed. This includes how overly naïve interpretation of how lipid exchange-induced changes in domain formation can impact protein function.

Structural and mechanistic insights into amyloid-ß and α-synuclein fibril formation and polyphenol inhibitor efficacy in phospholipid bilayers.

Under certain cellular conditions, functional proteins undergo misfolding, leading to a transition into oligomers which precede the formation of amyloid fibrils. Misfolding proteins are associated with neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. While the importance of lipid membranes in misfolding and disease aetiology is broadly accepted, the influence of lipid membranes during therapeutic design has been largely overlooked. This study utilized a biophysical approach to provide mechanistic insights into the effects of two lipid membrane systems (anionic and zwitterionic) on the inhibition of amyloid-ß 40 and α-synuclein amyloid formation at the monomer, oligomer and fibril level. Large unilamellar vesicles (LUVs) were shown to increase fibrillization and largely decrease the effectiveness of two well-known polyphenol fibril inhibitors, (-)-epigallocatechin gallate (EGCG) and resveratrol; however, use of immunoblotting and ion mobility mass spectrometry revealed this occurs through varying mechanisms. Oligomeric populations in particular were differentially affected by LUVs in the presence of resveratrol, an elongation phase inhibitor, compared to EGCG, a nucleation targeted inhibitor. Ion mobility mass spectrometry showed EGCG interacts with or induces more compact forms of monomeric protein typical of off-pathway structures; however, binding is reduced in the presence of LUVs, likely due to partitioning in the membrane environment. Competing effects of the lipids and inhibitor, along with reduced inhibitor binding in the presence of LUVs, provide a mechanistic understanding of decreased inhibitor efficacy in a lipid environment. Together, this study highlights that amyloid inhibitor design may be misguided if effects of lipid membrane composition and architecture are not considered during development.

α-Synuclein interacts differently with membranes mimicking the inner and outer leaflets of neuronal membranes.

The toxicity of α-synuclein (α-syn), the amyloidogenic protein responsible for Parkinson's disease, is likely related to its interaction with the asymmetric neuronal membrane. α-Syn exists as cytoplasmatic and as extracellular protein as well. To shed light on the different interactions occurring at the different α-syn localizations, we have here modelled the external and internal membrane leaflets of the neuronal membrane with two complex lipid mixtures, characterized by phase coexistence and with negative charge confined to either the ordered or the disordered phase, respectively. To this purpose, we selected a five-component (DOPC/SM/DOPE/DOPS/chol) and a four-component (DOPC/SM/GM1/chol) lipid mixtures, which contained the main membrane lipid constituents and exhibited a phase separation with formation of ordered domains. We have compared the action of α-syn in monomeric form and at different concentrations (1 nM, 40 nM, and 200 nM) with respect to lipid systems with different composition and shape by AFM, QCM-D, and vesicle leakage experiments. The experiments coherently showed a higher stability of the membranes composed by the internal leaflet mixture to the interaction with α-syn. Damage to membranes made of the external leaflet mixture was detected in a concentration-dependent manner. Interestingly, the membrane damage was related to the fluidity of the lipid domains and not to the presence of negatively charged lipids.

Bicontinuous inverted cubic phase stabilization as an index of antimicrobial and membrane fusion peptide activity.

Some antimicrobial peptides (AMPs) and membrane fusion-catalyzing peptides (FPs) stabilize bicontinuous inverted cubic (QII) phases. Previous authors proposed a topological rationale: since AMP-induced pores, fusion intermediates, and QII phases all have negative Gaussian curvature (NGC), peptides which produce NGC in one structure also do it in another. This assumes that peptides change the curvature energy of the lipid membranes. Here I test this with a Helfrich curvature energy model. First, experimentally, I show that lipid systems often used to study peptide NGC have NGC without peptides at higher temperatures. To determine the net effect of an AMP on NGC, the equilibrium phase behavior of the host lipids must be determined. Second, the model shows that AMPs must make large changes in the curvature energy to stabilize AMP-induced pores. Peptide-induced changes in elastic constants affect pores and QII phase differently. Changes in spontaneous curvature affect them in opposite ways. The observed correlation between QII phase stabilization and AMP activity doesn't show that AMPs act by lowering pore curvature energy. A different rationale is proposed. In theory, AMPs could simultaneously stabilize QII phase and pores by drastically changing two particular elastic constants. This could be tested by measuring AMP effects on the individual constants. I propose experiments to do that. Unlike AMPs, FPs must make only small changes in the curvature energy to catalyze fusion. It they act in this way, their fusion activity should correlate with their ability to stabilize QII phases.

Mutations in a barley cytochrome P450 gene enhances pathogen induced programmed cell death and cutin layer instability.

Disease lesion mimic mutants (DLMMs) are characterized by the spontaneous development of necrotic spots with various phenotypes designated as necrotic (nec) mutants in barley. The nec mutants were traditionally considered to have aberrant regulation of programmed cell death (PCD) pathways, which have roles in plant immunity and development. Most barley nec3 mutants express cream to orange necrotic lesions contrasting them from typical spontaneous DLMMs that develop dark pigmented lesions indicative of serotonin/phenolics deposition. Barley nec3 mutants grown under sterile conditions did not exhibit necrotic phenotypes until inoculated with adapted pathogens, suggesting that they are not typical DLMMs. The F2 progeny of a cross between nec3-γ1 and variety Quest segregated as a single recessive susceptibility gene post-inoculation with Bipolaris sorokiniana, the causal agent of the disease spot blotch. Nec3 was genetically delimited to 0.14 cM representing 16.5 megabases of physical sequence containing 149 annotated high confidence genes. RNAseq and comparative analysis of the wild type and five independent nec3 mutants identified a single candidate cytochrome P450 gene (HORVU.MOREX.r2.6HG0460850) that was validated as nec3 by independent mutations that result in predicted nonfunctional proteins. Histology studies determined that nec3 mutants had an unstable cutin layer that disrupted normal Bipolaris sorokiniana germ tube development.

Sphingomyelinase-Mediated Multitimescale Clustering of Ganglioside GM1 in Heterogeneous Lipid Membranes.

Several signaling processes in the plasma membrane are intensified by ceramides that are formed by sphingomyelinase-mediated hydrolysis of sphingomyelin. These ceramides trigger clustering of signaling-related biomolecules, but how they concentrate such biomolecules remains unclear. Here, the spatiotemporal localization of ganglioside GM1, a glycolipid receptor involved in signaling, during sphingomyelinase-mediated hydrolysis is described. Real-time visualization of the dynamic remodeling of the heterogeneous lipid membrane that occurs due to sphingomyelinase action is used to examine GM1 clustering, and unexpectedly, it is found that it is more complex than previously thought. Specifically, lipid membranes generate two distinct types of condensed GM1: 1) rapidly formed but short-lived GM1 clusters that are formed in ceramide-rich domains nucleated from the liquid-disordered phase; and 2) late-onset yet long-lasting, high-density GM1 clusters that are formed in the liquid-ordered phase. These findings suggest that multiple pathways exist in a plasma membrane to synergistically facilitate the rapid amplification and persistence of signals.

Crossing the lipid divide.

Archaeal membrane lipids are structurally different from bacterial and eukaryotic membrane lipids, but little is known about the enzymes involved in their synthesis. In a recent study, Exterkate et al. identified and characterized a cardiolipin synthase from the archaeon Methanospirillum hungatei. This enzyme can synthesize archaeal, bacterial, and mixed archaeal/bacterial cardiolipin species from a wide variety of substrates, some of which are not even naturally occurring. This discovery could revolutionize synthetic lipid biology, being used to construct a variety of lipids with nonnatural head groups and mixed archaeal/bacterial hydrophobic chains.

Mechanisms and functions of membrane lipid remodeling in plants.

Lipid remodeling, defined herein as post-synthetic structural modifications of membrane lipids, play crucial roles in regulating the physicochemical properties of cellular membranes and hence their many functions. Processes affected by lipid remodeling include lipid metabolism, membrane repair, cellular homeostasis, fatty acid trafficking, cellular signaling and stress tolerance. Glycerolipids are the major structural components of cellular membranes and their composition can be adjusted by modifying their head groups, their acyl chain lengths and the number and position of double bonds. This review summarizes recent advances in our understanding of mechanisms of membrane lipid remodeling with emphasis on the lipases and acyltransferases involved in the modification of phosphatidylcholine and monogalactosyldiacylglycerol, the major membrane lipids of extraplastidic and photosynthetic membranes, respectively. We also discuss the role of triacylglycerol metabolism in membrane acyl chain remodeling. Finally, we discuss emerging data concerning the functional roles of glycerolipid remodeling in plant stress responses. Illustrating the molecular basis of lipid remodeling may lead to novel strategies for crop improvement and other biotechnological applications such as bioenergy production.

Electron Paramagnetic Resonance Gives Evidence for the Presence of Type 1 Gonadotropin-Releasing Hormone Receptor (GnRH-R) in Subdomains of Lipid Rafts.

This study investigated the effect of type 1 gonadotropin releasing hormone receptor (GnRH-R) localization within lipid rafts on the properties of plasma membrane (PM) nanodomain structure. Confocal microscopy revealed colocalization of PM-localized GnRH-R with GM1-enriched raft-like PM subdomains. Electron paramagnetic resonance spectroscopy (EPR) of a membrane-partitioned spin probe was then used to study PM fluidity of immortalized pituitary gonadotrope cell line αT3-1 and HEK-293 cells stably expressing GnRH-R and compared it with their corresponding controls (αT4 and HEK-293 cells). Computer-assisted interpretation of EPR spectra revealed three modes of spin probe movement reflecting the properties of three types of PM nanodomains. Domains with an intermediate order parameter (domain 2) were the most affected by the presence of the GnRH-Rs, which increased PM ordering (order parameter (S)) and rotational mobility of PM lipids (decreased rotational correlation time (τc)). Depletion of cholesterol by methyl-ß-cyclodextrin (methyl-ß-CD) inhibited agonist-induced GnRH-R internalization and intracellular Ca2+ activity and resulted in an overall reduction in PM order; an observation further supported by molecular dynamics (MD) simulations of model membrane systems. This study provides evidence that GnRH-R PM localization may be related to a subdomain of lipid rafts that has lower PM ordering, suggesting lateral heterogeneity within lipid raft domains.

Pattern formation and polarity sorting of driven actin filaments on lipid membranes.

Collective motion of active matter is ubiquitously observed, ranging from propelled colloids to flocks of bird, and often features the formation of complex structures composed of agents moving coherently. However, it remains extremely challenging to predict emergent patterns from the binary interaction between agents, especially as only a limited number of interaction regimes have been experimentally observed so far. Here, we introduce an actin gliding assay coupled to a supported lipid bilayer, whose fluidity forces the interaction between self-propelled filaments to be dominated by steric repulsion. This results in filaments stopping upon binary collisions and eventually aligning nematically. Such a binary interaction rule results at high densities in the emergence of dynamic collectively moving structures including clusters, vortices, and streams of filaments. Despite the microscopic interaction having a nematic symmetry, the emergent structures are found to be polar, with filaments collectively moving in the same direction. This is due to polar biases introduced by the stopping upon collision, both on the individual filaments scale as well as on the scale of collective structures. In this context, positive half-charged topological defects turn out to be a most efficient trapping and polarity sorting conformation.

Effect of liver total sphingomyelin synthase deficiency on plasma lipid metabolism.

Sphingomyelin (SM) is one major phospholipids on lipoproteins. It is enriched on apolipoprotein B-containing particles, including very low-density lipoprotein (VLDL) and its catabolites, low-density lipoprotein (LDL). SM is synthesized by sphingomyelin synthase 1 and 2 (SMS1 and SMS2) which utilizes ceramide and phosphatidylcholine, as two substrates, to produce SM and diacylglyceride. SMS1 and SMS2 activities are co-expressed in all tested tissues, including the liver where VLDL is produced. Thus, neither Sms1 gene knockout (KO) nor Sms2 KO approach is sufficient to evaluate the effect of SMS on VLDL metabolism. We prepared liver-specific Sms1 KO/global Sms2 KO mice to evaluate the effect of hepatocyte SM biosynthesis in lipoprotein metabolism. We found that hepatocyte total SMS depletion significantly reduces cellular sphingomyelin levels. Also, we found that the deficiency induces cellular glycosphingolipid levels which is specifically related with SMS1 but not SMS2 deficiency. To our surprise, hepatocyte total SMS deficiency has marginal effect on hepatocyte ceramide, diacylglyceride, and phosphatidylcholine levels. Importantly, total SMS deficiency decreases plasma triglyceride but not apoB levels and reduces larger VLDL concentration. The reduction of triglyceride levels also was observed when the animals were on a high fat diet. Our results show that hepatocyte total SMS blocking can reduce VLDL-triglyceride production and plasma triglyceride levels. This phenomenon could be related with a reduction of atherogenicity.

Untargeted Lipidomics Analysis of the Cyanobacterium Synechocystis sp. PCC 6803: Lipid Composition Variation in Response to Alternative Cultivation Setups and to Gene Deletion.

Cyanobacteria play an important role in several ecological environments, and they are widely accepted to be the ancestors of chloroplasts in modern plants and green algae. Cyanobacteria have become attractive models for metabolic engineering, with the goal of exploring them as microbial cell factories. However, the study of cyanobacterial lipids' composition and variation, and the assessment of the lipids' functional and structural roles have been largely overlooked. Here, we aimed at expanding the cyanobacterial lipidomic analytical pipeline by using an untargeted lipidomics approach. Thus, the lipid composition variation of the model cyanobacterium Synechocystis sp. PCC 6803 was investigated in response to both alternative cultivation setups and gene deletion. This approach allowed for detecting differences in total lipid content, alterations in fatty-acid unsaturation level, and adjustments of specific lipid species among the identified lipid classes. The employed method also revealed that the cultivation setup tested in this work induced a deeper alteration of the cyanobacterial cell lipidome than the deletion of a gene that results in a dramatic increase in the release of lipid-rich outer membrane vesicles. This study further highlights how growth conditions must be carefully selected when cyanobacteria are to be engineered and/or scaled-up for lipid or fatty acids production.

Effects of ATP9A on Extracellular Vesicle Release and Exosomal Lipid Composition.

Numerous biological processes are regulated by the intercellular communications arising from extracellular vesicles (EVs) released from cells. However, the mechanisms that regulate the quantity of EV discharged have yet to be understood. While it is known that ATP9A, a P4-ATPase, is involved in endosomal recycling, it is not clear whether it also contributes to the release of EVs and the makeup of exosomal lipids. This study is aimed at exploring the role of human ATP9A in the process of EV release and, further, to analyze the profiles of EV lipids regulated by ATP9A. Our results demonstrate that ATP9A is located in both the intracellular compartments and the plasma membrane. The percentage of ceramides and sphingosine was found to be significantly greater in the control cells than in the ATP9A overexpression and ATP9A knockout groups. However, EV release was greater in ATP9A knockout cells, indicating that ATP9A inhibits the release of EVs. This study revealed the effects of ATP9A on the release of EVs and the lipid composition of exosomes.

Genetically controlled membrane synthesis in liposomes.

Lipid membranes, nucleic acids, proteins, and metabolism are essential for modern cellular life. Synthetic systems emulating the fundamental properties of living cells must therefore be built upon these functional elements. In this work, phospholipid-producing enzymes encoded in a synthetic minigenome are cell-free expressed within liposome compartments. The de novo synthesized metabolic pathway converts precursors into a variety of lipids, including the constituents of the parental liposome. Balanced production of phosphatidylethanolamine and phosphatidylglycerol is realized, owing to transcriptional regulation of the activity of specific genes combined with a metabolic feedback mechanism. Fluorescence-based methods are developed to image the synthesis and membrane incorporation of phosphatidylserine at the single liposome level. Our results provide experimental evidence for DNA-programmed membrane synthesis in a minimal cell model. Strategies are discussed to alleviate current limitations toward effective liposome growth and self-reproduction.

Deciphering the Novel Role of AtMIN7 in Cuticle Formation and Defense against the Bacterial Pathogen Infection.

The cuticle is the outermost layer of plant aerial tissue that interacts with the environment and protects plants against water loss and various biotic and abiotic stresses. ADP ribosylation factor guanine nucleotide exchange factor proteins (ARF-GEFs) are key components of the vesicle trafficking system. Our study discovers that AtMIN7, an Arabidopsis ARF-GEF, is critical for cuticle formation and related leaf surface defense against the bacterial pathogen Pseudomonas syringae pathovar tomato (Pto). Our transmission electron microscopy and scanning electron microscopy studies indicate that the atmin7 mutant leaves have a thinner cuticular layer, defective stomata structure, and impaired cuticle ledge of stomata compared to the leaves of wild type plants. GC-MS analysis further revealed that the amount of cutin monomers was significantly reduced in atmin7 mutant plants. Furthermore, the exogenous application of either of three plant hormones-salicylic acid, jasmonic acid, or abscisic acid-enhanced the cuticle formation in atmin7 mutant leaves and the related defense responses to the bacterial Pto infection. Thus, transport of cutin-related components by AtMIN7 may contribute to its impact on cuticle formation and related defense function.

Hepatitis C virus NS3-4A protease regulates the lipid environment for RNA replication by cleaving host enzyme 24-dehydrocholesterol reductase.

Many RNA viruses create specialized membranes for genome replication by manipulating host lipid metabolism and trafficking, but in most cases, we do not know the molecular mechanisms responsible or how specific lipids may impact the associated membrane and viral process. For example, hepatitis C virus (HCV) causes a specific, large-fold increase in the steady-state abundance of intracellular desmosterol, an immediate precursor of cholesterol, resulting in increased fluidity of the membrane where HCV RNA replication occurs. Here, we establish the mechanism responsible for HCV's effect on intracellular desmosterol, whereby the HCV NS3-4A protease controls activity of 24-dehydrocholesterol reductase (DHCR24), the enzyme that catalyzes conversion of desmosterol to cholesterol. Our cumulative evidence for the proposed mechanism includes immunofluorescence microscopy experiments showing co-occurrence of DHCR24 and HCV NS3-4A protease; formation of an additional, faster-migrating DHCR24 species (DHCR24*) in cells harboring a HCV subgenomic replicon RNA or ectopically expressing NS3-4A; and biochemical evidence that NS3-4A cleaves DHCR24 to produce DHCR24* in vitro and in vivo We further demonstrate that NS3-4A cleaves DHCR24 between residues Cys91 and Thr92 and show that this reduces the intracellular conversion of desmosterol to cholesterol. Together, these studies demonstrate that NS3-4A directly cleaves DHCR24 and that this results in the enrichment of desmosterol in the membranes where NS3-4A and DHCR24 co-occur. Overall, this suggests a model in which HCV directly regulates the lipid environment for RNA replication through direct effects on the host lipid metabolism.

Membrane Lipid Requirements of the Lysine Transporter Lyp1 from Saccharomyces cerevisiae.

Membrane lipids act as solvents and functional cofactors for integral membrane proteins. The yeast plasma membrane is unusual in that it may have a high lipid order, which coincides with low passive permeability for small molecules and a slow lateral diffusion of proteins. Yet, membrane proteins whose functions require altered conformation must have flexibility within membranes. We have determined the molecular composition of yeast plasma membrane lipids located within a defined diameter of model proteins, including the APC-superfamily lysine transporter Lyp1. We now use the composition of lipids that naturally surround Lyp1 to guide testing of lipids that support the normal functioning of the transporter, when reconstituted in vesicles of defined lipid composition. We find that phosphatidylserine and ergosterol are essential for Lyp1 function, and the transport activity displays a sigmoidal relationship with the concentration of these lipids. Non-bilayer lipids stimulate transport activity, but different types are interchangeable. Remarkably, Lyp1 requires a relatively high fraction of lipids with one or more unsaturated acyl chains. The transport data and predictions of the periprotein lipidome of Lyp1 support a new model in which a narrow band of lipids immediately surrounding the transmembrane stalk of a model protein allows conformational changes in the protein.