Carrier-Tumor Cell Membrane Interactions for Optimized Delivery of a Promising Drug, 4(RS)-4-F4t-Neuroprostane.

Nanomedicines engineered to deliver molecules with therapeutic potentials, overcoming drawbacks such as poor solubility, toxicity or a short half-life, are targeted towards their cellular destination either passively or through various elements of cell membranes. The differences in the physicochemical properties of the cell membrane between tumor and nontumor cells have been reported, but they are not systematically used for drug delivery purposes. Thus, in this study, a new approach based on a match between the liposome compositions, i.e., membrane fluidity, to selectively interact with the targeted cell membrane was used. Lipid-based carriers of two different fluidities were designed and used to deliver 4(RS)-4-F4t-Neuroprostane (F4t-NeuroP), a potential antitumor molecule derived from docosahexaenoic acid (DHA). Based on its hydrophobic character, F4t-NeuroP was added to the lipid mixture prior to liposome formation, a protocol that yielded over 80% encapsulation efficiency in both rigid and fluid liposomes. The presence of the active molecule did not modify the liposome size but increased the liposome negative charge and the liposome membrane fluidity, which suggested that the active molecule was accommodated in the lipid membrane. F4t-NeuroP integration in liposomes with a fluid character allowed for the selective targeting of the metastatic prostate cell line PC-3 vs. fibroblast controls. A significant decrease in viability (40%) was observed for the PC-3 cancer line in the presence of F4t-NeuroP fluid liposomes, whereas rigid F4t-NeuroP liposomes did not alter the PC-3 cell viability. These findings demonstrate that liposomes encapsulating F4t-NeuroP or other related molecules may be an interesting model of drug carriers based on membrane fluidity.

Morphological and Mechanical Characterization of Extracellular Vesicles and Parent Human Synoviocytes under Physiological and Inflammatory Conditions.

The morphology of fibroblast-like synoviocytes (FLS) issued from the synovial fluid (SF) of patients suffering from osteoarthritis (OA), rheumatoid arthritis (RA), or from healthy subjects (H), as well as the ultrastructure and mechanical properties of the FLS-secreted extracellular vesicles (EV), were analyzed by confocal microscopy, transmission electron microscopy, atomic force microscopy, and tribological tests. EV released under healthy conditions were constituted of several lipid bilayers surrounding a viscous inner core. This "gel-in" vesicular structure ensured high mechanical resistance of single vesicles and good tribological properties of the lubricant. RA, and to a lesser extent OA, synovial vesicles had altered morphology, corresponding to a "gel-out" situation with vesicles surrounded by a viscous gel, poor mechanical resistance, and poor lubricating qualities. When subjected to inflammatory conditions, healthy cells developed phenotypes similar to that of RA samples, which reinforces the importance of inflammatory processes in the loss of lubricating properties of SF.

Monomethyl Auristatin E Grafted-Liposomes to Target Prostate Tumor Cell Lines.

Novel nanomedicines have been engineered to deliver molecules with therapeutic potentials, overcoming drawbacks such as poor solubility, toxicity or short half-life. Lipid-based carriers such as liposomes represent one of the most advanced classes of drug delivery systems. A Monomethyl Auristatin E (MMAE) warhead was grafted on a lipid derivative and integrated in fusogenic liposomes, following the model of antibody drug conjugates. By modulating the liposome composition, we designed a set of particles characterized by different membrane fluidities as a key parameter to obtain selective uptake from fibroblast or prostate tumor cells. Only the fluid liposomes made of palmitoyl-oleoyl-phosphatidylcholine and dioleoyl-phosphatidylethanolamine, integrating the MMAE-lipid derivative, showed an effect on prostate tumor PC-3 and LNCaP cell viability. On the other hand, they exhibited negligible effects on the fibroblast NIH-3T3 cells, which only interacted with rigid liposomes. Therefore, fluid liposomes grafted with MMAE represent an interesting example of drug carriers, as they can be easily engineered to promote liposome fusion with the target membrane and ensure drug selectivity.

Membrane Fluidity as a New Means to Selectively Target Cancer Cells with Fusogenic Lipid Carriers.

Lipid-based carriers such as liposomes represent one of the most advanced classes of drug delivery systems. Their clinical success relies on their composition, similar to that of the cell membrane. Their cellular specificity often relies on a ligand-receptor interaction. Although differences in the physicochemical properties of the cell membrane between tumor and nontumor cells have been reported, they are not systematically used for drug delivery purposes. In this report, a new approach was developed to ensure selective targeting based on physical compatibility between the target and the carrier membranes. By modulating the liposome composition and thus its membrane fluidity, we achieved selective targeting on four cancer cell lines of varying aggressiveness. Furthermore, using membrane-embedded and inner core-encapsulated fluorophores, we assessed the mechanism of this interaction to be based on the fusion of the liposome with the cell membranes. Membrane fluidity is therefore a major parameter to be considered when designing lipid drug carriers as a promising, lower cost alternative to current targeting strategies based on covalent grafting.

Mechanism of activation of plant monogalactosyldiacylglycerol synthase 1 (MGD1) by phosphatidylglycerol.

Mono- and digalactosyldiacylglycerol are essential galactolipids for the biogenesis of plastids and functioning of the photosynthetic machinery. In Arabidopsis, the first step of galactolipid synthesis is catalyzed by monogalactosyldiacylglycerol synthase 1 (MGD1), a monotopic protein located in the inner envelope membrane of chloroplasts, which transfers a galactose residue from UDP-galactose to diacylglycerol (DAG). MGD1 needs anionic lipids such as phosphatidylglycerol (PG) to be active, but the mechanism by which PG activates MGD1 is still unknown. Recent studies shed light on the catalytic mechanism of MGD1 and on the possible PG binding site. Particularly, Pro189 was identified as a potential residue implicated in PG binding and His155 as the putative catalytic residue. In the present study, using a multifaceted approach (Langmuir membrane models, atomic force microscopy, molecular dynamics; MD), we investigated the membrane binding properties of native MGD1 and mutants (P189A and H115A). We demonstrated that both residues are involved in PG binding, thus suggesting the existence of a PG-His catalytic dyad that should facilitate deprotonation of the nucleophile hydroxyl group of DAG acceptor. Interestingly, MD simulations showed that MGD1 induces a reorganization of lipids by attracting DAG molecules to create an optimal platform for binding.

Quantitative atomic force microscopy provides new insight into matrix vesicle mineralization.

Matrix vesicles (MVs) are a class of extracellular vesicles that initiate mineralization in cartilage, bone, and other vertebrate tissues by accumulating calcium ions (Ca2+) and inorganic phosphate (Pi) within their lumen and forming a nucleation core (NC). After further sequestration of Ca2+ and Pi, the NC transforms into crystalline complexes. Direct evidence of the existence of the NC and its maturation have been provided solely by analyses of dried samples. We isolated MVs from chicken embryo cartilage and used atomic force microscopy peak force quantitative nanomechanical property mapping (AFM-PFQNM) to measure the nanomechanical and morphological properties of individual MVs under both mineralizing (+Ca2+) and non-mineralizing (-Ca2+) fluid conditions. The elastic modulus of MVs significantly increased by 4-fold after incubation in mineralization buffer. From AFM mapping data, we inferred the morphological changes of MVs as mineralization progresses: prior to mineralization, a punctate feature, the NC, is present within MVs and this feature grows and stiffens during mineralization until it occupies most of the MV lumen. Dynamic light scattering showed a significant increase in hydrodynamic diameter and no change in the zeta potential of hydrated MVs after incubation with Ca2+. This validates that crystalline complexes, which are strongly negative relative to MVs, were forming within the lumen of MVs. These data were substantiated by transmission electron microscopy energy dispersive X-ray and Fourier transform infrared spectroscopic analyses of dried MVs, which provide evidence that the complexes increased in size, crystallinity, and Ca/P ratio within MVs during the mineralization process.

A new functional membrane protein microarray based on tethered phospholipid bilayers.

A new prototype of a membrane protein biochip is presented in this article. This biochip was created by the combination of novel technologies of peptide-tethered bilayer lipid membrane (pep-tBLM) formation and solid support micropatterning. Pep-tBLMs integrating a membrane protein were obtained in the form of microarrays on a gold chip. The formation of the microspots was visualized in real-time by surface plasmon resonance imaging (SPRi) and the functionality of a GPCR (CXCR4), reinserted locally into microwells, was assessed by ligand binding studies. In brief, to achieve micropatterning, P19-4H, a 4 histidine-possessing peptide spacer, was spotted inside microwells obtained on polystyrene-coated gold, and Ni-chelating proteoliposomes were injected into the reaction chamber. Proteoliposome binding to the peptide was based on metal-chelate interaction. The peptide-tethered lipid bilayer was finally obtained by addition of a fusogenic peptide (AH peptide) to promote proteoliposome fusion. The CXCR4 pep-tBLM microarray was characterized by surface plasmon resonance imaging (SPRi) throughout the building-up process. This new generation of membrane protein biochip represents a promising method of developing a screening tool for drug discovery.

Do Galactolipid Synthases Play a Key Role in the Biogenesis of Chloroplast Membranes of Higher Plants?

A unique feature of chloroplasts is their high content of the galactolipids monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), which constitute up to 80% of their lipids. These galactolipids are synthesized in the chloroplast envelope membrane through the concerted action of galactosyltransferases, the so-called 'MGDG synthases (MGDs)' and 'DGDG synthases (DGDs),' which use uridine diphosphate (UDP)-galactose as donor. In Arabidopsis leaves, under standard conditions, the enzymes MGD1 and DGD1 provide the bulk of galactolipids, necessary for the massive expansion of thylakoid membranes. Under phosphate limited conditions, plants activate another pathway involving MGD2/MGD3 and DGD2 to provide additional DGDG that is exported to extraplastidial membranes where they partly replace phospholipids, a phosphate-saving mechanism in plants. A third enzyme system, which relies on the UDP-Gal-independent GGGT (also called SFR2 for SENSITIVE TO FREEZING 2), can be activated in response to a freezing stress. The biosynthesis of galactolipids by these multiple enzyme sets must be tightly regulated to meet the cellular demand in response to changing environmental conditions. The cooperation between MGD and DGD enzymes with a possible substrate channeling from diacylglycerol to MGDG and DGDG is supported by biochemical and biophysical studies and mutant analyses reviewed herein. The fine-tuning of MGDG to DGDG ratio, which allows the reversible transition from the hexagonal II to lamellar α phase of the lipid bilayer, could be a key factor in thylakoid biogenesis.

The potent effect of mycolactone on lipid membranes.

Mycolactone is a lipid-like endotoxin synthesized by an environmental human pathogen, Mycobacterium ulcerans, the causal agent of Buruli ulcer disease. Mycolactone has pleiotropic effects on fundamental cellular processes (cell adhesion, cell death and inflammation). Various cellular targets of mycolactone have been identified and a literature survey revealed that most of these targets are membrane receptors residing in ordered plasma membrane nanodomains, within which their functionalities can be modulated. We investigated the capacity of mycolactone to interact with membranes, to evaluate its effects on membrane lipid organization following its diffusion across the cell membrane. We used Langmuir monolayers as a cell membrane model. Experiments were carried out with a lipid composition chosen to be as similar as possible to that of the plasma membrane. Mycolactone, which has surfactant properties, with an apparent saturation concentration of 1 µM, interacted with the membrane at very low concentrations (60 nM). The interaction of mycolactone with the membrane was mediated by the presence of cholesterol and, like detergents, mycolactone reshaped the membrane. In its monomeric form, this toxin modifies lipid segregation in the monolayer, strongly affecting the formation of ordered microdomains. These findings suggest that mycolactone disturbs lipid organization in the biological membranes it crosses, with potential effects on cell functions and signaling pathways. Microdomain remodeling may therefore underlie molecular events, accounting for the ability of mycolactone to attack multiple targets and providing new insight into a single unifying mechanism underlying the pleiotropic effects of this molecule. This membrane remodeling may act in synergy with the other known effects of mycolactone on its intracellular targets, potentiating these effects.

Glass Microsphere-Supported Giant Vesicles for the Observation of Self-Reproduction of Lipid Boundaries.

Growth and division experiments on phospholipid boundaries were carried out using glass microsphere-supported phospholipid (DOPC) giant vesicles (GVs) fed with a fatty acid solution (oleic acid) at two distinct feeding rates. Both fast and slow feeding methods produced daughter GVs. Under slow feeding conditions the membrane growth process (evagination, buds, filaments) was observed in detail by fluorescence microscopy. The density difference between supported mother vesicles and newly formed daughter vesicles allowed their easy separation. Mass spectrometric analysis of the resulting mother and daughter GVs showed that the composition of both vesicle types was a mixture of original supported phospholipids and added fatty acids reflecting the total composition of amphiphiles after the feeding process. Thus, self-reproduction of phospholipid vesicles can take place under preservation of the lipid composition but different aggregate size.

Interfacial Properties of NTAIL, an Intrinsically Disordered Protein.

Intrinsically disordered proteins (IDPs) lack stable secondary and tertiary structure under physiological conditions in the absence of their biological partners and thus exist as dynamic ensembles of interconverting conformers, often highly soluble in water. However, in some cases, IDPs such as the ones involved in neurodegenerative diseases can form protein aggregates and their aggregation process may be triggered by the interaction with membranes. Although the interfacial behavior of globular proteins has been extensively studied, experimental data on IDPs at the air/water (A/W) and water/lipid interfaces are scarce. We studied here the intrinsically disordered C-terminal domain of the Hendra virus nucleoprotein (NTAIL) and compared its interfacial properties to those of lysozyme that is taken as a model globular protein of similar molecular mass. Adsorption of NTAIL at the A/W interface was studied in the absence and presence of phospholipids using Langmuir films, polarization modulated-infrared reflection-absorption spectroscopy, and an automated drop tensiometer for interfacial tension and elastic modulus determination with oscillating bubbles. NTAIL showed a significant surface activity, with a higher adsorption capacity at the A/W interface and penetration into egg phosphatidylcholine monolayer compared to lysozyme. Whereas lysozyme remains folded upon compression of the protein layer at the A/W interface and shows a quasi-pure elastic behavior, NTAIL shows a much higher molecular area and forms a highly viscoelastic film with a high dilational modulus. To our knowledge, a new disorder-to-order transition is thus observed for the NTAIL protein that folds into an antiparallel ß-sheet at the A/W interface and presents strong intermolecular interactions.

New Tethered Phospholipid Bilayers Integrating Functional G-Protein-Coupled Receptor Membrane Proteins.

Membrane proteins exhibiting extra- and intracellular domains require an adequate near-native lipid platform for their functional reconstitution. With this aim, we developed a new technology enabling the formation of a peptide-tethered bilayer lipid membrane (pep-tBLM), a lipid bilayer grafted onto peptide spacers, by way of a metal-chelate interaction. To this end, we designed an original peptide spacer derived from the natural α-laminin thiopeptide (P19) possessing a cysteine residue in the N-terminal extremity for grafting onto gold and a C-terminal extremity modified by four histidine residues (P19-4H). In the presence of nickel, the use of this anchor allowed us to bind liposomes of variable compositions containing a 2% molar ratio of a chelating lipid, 1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] so-called DOGS-NTA, and to form the planar bilayer by triggering liposome fusion by an α-helical (AH) peptide derived from the N-terminus of the hepatitis C virus NS5A protein. The formation of pep-tBLMs was characterized by surface plasmon resonance imaging (SPRi), and their continuity, fluidity, and homogeneity were demonstrated by fluorescence recovery after photobleaching (FRAP), with a diffusion coefficient of 2.5 × 10-7 cm2/s, and atomic force microscopy (AFM). By using variable lipid compositions including phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylinositol 4,5-bisphosphate (PIP2), sphingomyelin (SM), phosphatidic acid (PA), and cholesterol (Chol) in various ratios, we show that the membrane can be formed independently from the lipid composition. We made the most of this advantage to reincorporate a transmembrane protein in an adapted complex lipid composition to ensure its functional reinsertion. For this purpose, a cell-free expression system was used to produce proteoliposomes expressing the functional C-X-C motif chemokine receptor 4 (CXCR4), a seven-transmembrane protein belonging to the large superfamily of G-protein-coupled receptors (GPCRs). We succeeded in reinserting CXCR4 in pep-tBLMs formed on P19-4H by the fusion of tethered proteoliposomes. AFM and FRAP characterization allowed us to show that pep-tBLMs inserting CXCR4 remained fluid, homogeneous, and continuous. The value of the diffusion coefficient determined in the presence of reinserted CXCR4 was 2 × 10-7 cm2/s. Ligand binding assays using a synthetic CXCR4 antagonist, T22 ([Tyr5,12, Lys7]-polyphemusin II), revealed that CXCR4 can be reinserted in pep-tBLMs with functional folding and orientation. This new approach represents a method of choice for investigating membrane protein reincorporation and a promising way of creating a new generation of membrane biochips adapted for screening agonists or antagonists of transmembrane proteins.

Correction: Giant vesicles from rehydrated crude mixtures containing unexpected mixtures of amphiphiles formed under plausibly prebiotic conditions.

Correction for 'Giant vesicles from rehydrated crude mixtures containing unexpected mixtures of amphiphiles formed under plausibly prebiotic conditions' by Michele Fiore et al., Org. Biomol. Chem., 2017, 15, 4231-4240.

Giant vesicles from rehydrated crude mixtures containing unexpected mixtures of amphiphiles formed under plausibly prebiotic conditions.

Giant lipid vesicles resemble compartments of biological cells, mimicking them in their dimension, membrane structure and partly in their membrane composition. The spontanenous appearance of closed membranes composed of bilayers of self-assembling amphiphiles was likely a prerequisite for Darwinian competitive behavior to set in at the molecular level. Such compartments should be dynamic in their membrane composition (evolvable), and sufficiently stable to harbor macromolecules (leak-free), yet semi-permeable for reactive small molecules to get across the membrane (stay away from chemical equilibrium). Here we describe bottom-up experiments simulating prebiotic environments that support the formation of simple amphiphilic molecules capable of self-assembling into vesicular objects on the micrometer scale. Long-chain alkyl phosphates, together with related amphiphilic compounds, were formed under simulated prebiotic phosphorylation conditions by using cyanamide, a recognized prebiotic chemical activator and a precursor for several compound classes. Crude dry material of the thus obtained prebiotic mixtures formed multilamellar giant vesicles once rehydrated at the appropriate pH and in the presence of plausibly prebiotic co-surfactants, as observed by optical microscopy. The size and the shape of lipid aggregates tentatively suggest that prebiotic lipid assemblies could encapsulate peptides or nucleic acids that could be formed under similar chemical prebiotic conditions. The formation of prebiotic amphiphiles was monitored by using TLC, IR, NMR and ESI-MS and UPLC-HRMS. In addition we provide a spectroscopic analysis of cyanamide under simulated prebiotic conditions in the presence of phosphate sources and spectroscopic analysis of O-phosphorylethanolamine as a plausible precursor for phosphoethanolamine lipids.

Structural insights and membrane binding properties of MGD1, the major galactolipid synthase in plants.

Monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) are the major lipid components of photosynthetic membranes, and hence the most abundant lipids in the biosphere. They are essential for assembly and function of the photosynthetic apparatus. In Arabidopsis, the first step of galactolipid synthesis is catalyzed by MGDG synthase 1 (MGD1), which transfers a galactosyl residue from UDP-galactose to diacylglycerol (DAG). MGD1 is a monotopic protein that is embedded in the inner envelope membrane of chloroplasts. Once produced, MGDG is transferred to the outer envelope membrane, where DGDG synthesis occurs, and to thylakoids. Here we present two crystal structures of MGD1: one unliganded and one complexed with UDP. MGD1 has a long and flexible region (approximately 50 amino acids) that is required for DAG binding. The structures reveal critical features of the MGD1 catalytic mechanism and its membrane binding mode, tested on biomimetic Langmuir monolayers, giving insights into chloroplast membrane biogenesis. The structural plasticity of MGD1, ensuring very rapid capture and utilization of DAG, and its interaction with anionic lipids, possibly driving the construction of lipoproteic clusters, are consistent with the role of this enzyme, not only in expansion of the inner envelope membrane, but also in supplying MGDG to the outer envelope and nascent thylakoid membranes.

New Insights on Thylakoid Biogenesis in Plant Cells.

Photosynthetic membranes, or thylakoids, are the most extensive membrane system found in the biosphere. They form flattened membrane cisternae in the cytosol of cyanobacteria and in the stroma of chloroplasts. The efficiency of light energy capture and conversion, critical for primary production in ecosystems, relies on the rapid expansion of thylakoids and their versatile reorganization in response to light changes. Thylakoid biogenesis results from the assembly of a lipid matrix combined with the incorporation of protein components. Four lipid classes are conserved from cyanobacteria to chloroplasts: mono- and digalactosyldiacylglycerol, sulfoquinovosyldiacylglycerol, and phosphatidyldiacylglycerol. This review focuses on the production and biophysical properties of galactolipids, making them determinant factors for the nonvesicular/nonlamellar biogenesis and for the three-dimensional architecture of nascent thylakoids. The regulation of MGD1, the committing enzyme of galactolipid biosynthesis in Arabidopsis, via feedback regulatory loops and control of protein binding to membranes, is also detailed.

Carrier-inside-carrier: polyelectrolyte microcapsules as reservoir for drug-loaded liposomes.

Conventional liposomes have a short life-time in blood, unless they are protected by a polymer envelope, most often polyethylene glycol. However, these stabilizing polymers frequently interfere with cellular uptake, impede liposome-membrane fusion and inhibit escape of liposome content from endosomes. To overcome such drawbacks, polymer-based systems as carriers for liposomes are currently developed. Conforming to this approach, we propose a new and convenient method for embedding small size liposomes, 30-100 nm, inside porous calcium carbonate microparticles. These microparticles served as templates for deposition of various polyelectrolytes to form a protective shell. The carbonate particles were then dissolved to yield hollow polyelectrolyte microcapsules. The main advantage of using this method for liposome encapsulation is that carbonate particles can serve as a sacrificial template for deposition of virtually any polyelectrolyte. By carefully choosing the shell composition, bioavailability of the liposomes and of the encapsulated drug can be modulated to respond to biological requirements and to improve drug delivery to the cytoplasm and avoid endosomal escape.

Tethered bilayer lipid membranes (tBLMs): interest and applications for biological membrane investigations.

Biological membranes play a central role in the biology of the cell. They are not only the hydrophobic barrier allowing separation between two water soluble compartments but also a supra-molecular entity that has vital structural functions. Notably, they are involved in many exchange processes between the outside and inside cellular spaces. Accounting for the complexity of cell membranes, reliable models are needed to acquire current knowledge of the molecular processes occurring in membranes. To simplify the investigation of lipid/protein interactions, the use of biomimetic membranes is an approach that allows manipulation of the lipid composition of specific domains and/or the protein composition, and the evaluation of the reciprocal effects. Since the middle of the 80's, lipid bilayer membranes have been constantly developed as models of biological membranes with the ultimate goal to reincorporate membrane proteins for their functional investigation. In this review, after a brief description of the planar lipid bilayers as biomimetic membrane models, we will focus on the construction of the tethered Bilayer Lipid Membranes, the most promising model for efficient membrane protein reconstitution and investigation of molecular processes occurring in cell membranes.

Specific interaction to PIP2 increases the kinetic rate of membrane binding of VILIPs, a subfamily of Neuronal Calcium Sensors (NCS) proteins.

VIsinin-LIke Proteins (VILIPs) are a subfamily of the Neuronal Calcium Sensor (NCS) proteins, which possess both N-myristoylation and EF-hand motifs allowing for a putative 'calcium-myristoyl switch' regulation mechanism. It has previously been established that myristoyl conjugation increases the affinity of proteins for membranes, but, in many cases, a second feature such as a cluster of positively-charged residues is needed for stable membrane binding. The interaction of two members of this family, VILIP-1 and VILIP-3, with Langmuir monolayers as membrane models has been investigated in order to study the effects of both myristoylation and the highly basic region containing conserved poly-lysine residues on membrane association kinetics and binding properties. Results show that in the presence of calcium, N-myristoylation significantly increases the kinetic rate of VILIP adsorption to the membrane. Additionally, the proteins bind to negatively charged phospholipids independently of the conjugated myristate moiety. Besides the regulatory effect of calcium on the rate of binding presumably due to exposure of the myristoyl moiety ascribed to their putative 'calcium-myristoyl switch', VILIP-1 and -3 also engage specific interactions with biomimetic membranes containing phosphatidylinositol 4,5-bisphosphate (PIP2). The presence of PIP2 increases the membrane association rates of both VILIPs. Taken together, these results show the major kinetic role of N-myristoylation for membrane binding, and highlight the critical role of specific phosphoinositide interactions for membrane association of members of the VILIP family.

The influence of lipids on MGD1 membrane binding highlights novel mechanisms for galactolipid biosynthesis regulation in chloroplasts.

Mono- and digalactosyldiacylglycerol (MGDG and DGDG) are the most abundant lipids of photosynthetic membranes (thylakoids). In Arabidopsis green tissues, MGD1 is the main enzyme synthesizing MGDG. This monotopic enzyme is embedded in the inner envelope membrane of chloroplasts. DGDG synthesis occurs in the outer envelope membrane. Although the suborganellar localization of MGD1 has been determined, it is still not known how the lipid/glycolipid composition influences its binding to the membrane. The existence of a topological relationship between MGD1 and "embryonic" thylakoids is also unknown. To investigate MGD1 membrane binding, we used a Langmuir membrane model allowing the tuning of both lipid composition and packing. Surprisingly, MGD1 presents a high affinity to MGDG, its product, which maintains the enzyme bound to the membrane. This positive feedback is consistent with the low level of diacylglycerol, the substrate of MGD1, in chloroplast membranes. By contrast, MGD1 is excluded from membranes highly enriched in, or made of, pure DGDG. DGDG therefore exerts a retrocontrol, which is effective on the overall synthesis of galactolipids. Previously identified activators, phosphatidic acid and phosphatidylglycerol, also play a role on MGD1 membrane binding via electrostatic interactions, compensating the exclusion triggered by DGDG. The opposite effects of MGDG and DGDG suggest a role of these lipids on the localization of MGD1 in specific domains. Consistently, MGDG induces the self-organization of MGD1 into elongated and reticulated nanostructures scaffolding the chloroplast membrane.-Sarkis, J., Rocha, J., Maniti, O., Jouhet, J., Vié, V., Block, M. A., Breton, C., Maréchal, E., Girard-Egrot, A. The influence of lipids on MGD1 membrane binding highlights novel mechanisms for galactolipid biosynthesis regulation in chloroplasts.