Biophysical analysis of the plant-specific GIPC sphingolipids reveals multiple modes of membrane regulation.

The plant plasma membrane (PM) is an essential barrier between the cell and the external environment, controlling signal perception and transmission. It consists of an asymmetrical lipid bilayer made up of three different lipid classes: sphingolipids, sterols, and phospholipids. The glycosyl inositol phosphoryl ceramides (GIPCs), representing up to 40% of total sphingolipids, are assumed to be almost exclusively in the outer leaflet of the PM. However, their biological role and properties are poorly defined. In this study, we investigated the role of GIPCs in membrane organization. Because GIPCs are not commercially available, we developed a protocol to extract and isolate GIPC-enriched fractions from eudicots (cauliflower and tobacco) and monocots (leek and rice). Lipidomic analysis confirmed the presence of trihydroxylated long chain bases and 2-hydroxylated very long-chain fatty acids up to 26 carbon atoms. The glycan head groups of the GIPCs from monocots and dicots were analyzed by gas chromatograph-mass spectrometry, revealing different sugar moieties. Multiple biophysics tools, namely Langmuir monolayer, ζ-Potential, light scattering, neutron reflectivity, solid state 2H-NMR, and molecular modeling, were used to investigate the physical properties of the GIPCs, as well as their interaction with free and conjugated phytosterols. We showed that GIPCs increase the thickness and electronegativity of model membranes, interact differentially with the different phytosterols species, and regulate the gel-to-fluid phase transition during temperature variations. These results unveil the multiple roles played by GIPCs in the plant PM.

Lysophosphatidic acid acyltransferases: a link with intracellular protein trafficking in Arabidopsis root cells?

Phosphatidic acid (PA) and lysophosphatidic acid acyltransferases (LPAATs) might be critical for the secretory pathway. Four extra-plastidial LPAATs (LPAAT2, 3, 4, and 5) were identified in Arabidopsis thaliana. These AtLPAATs display a specific enzymatic activity converting lysophosphatidic acid to PA and are located in the endomembrane system. We investigate a putative role for AtLPAATs 3, 4, and 5 in the secretory pathway of root cells through genetical (knockout mutants), biochemical (activity inhibitor, lipid analyses), and imaging (live and immuno-confocal microscopy) approaches. Treating a lpaat4;lpaat5 double mutant with the LPAAT inhibitor CI976 produced a significant decrease in primary root growth. The trafficking of the auxin transporter PIN2 was disturbed in this lpaat4;lpaat5 double mutant treated with CI976, whereas trafficking of H+-ATPases was unaffected. The lpaat4;lpaat5 double mutant is sensitive to salt stress, and the trafficking of the aquaporin PIP2;7 to the plasma membrane in the lpaat4;lpaat5 double mutant treated with CI976 was reduced. We measured the amounts of neo-synthesized PA in roots, and found a decrease in PA only in the lpaat4;lpaat5 double mutant treated with CI976, suggesting that the protein trafficking impairment was due to a critical PA concentration threshold.

Doxorubicin Inhibits Phosphatidylserine Decarboxylase and Modifies Mitochondrial Membrane Composition in HeLa Cells.

Doxorubicin (DXR) is a drug widely used in chemotherapy. Its mode of action is based on its intercalation properties, involving the inhibition of topoisomerase II. However, few studies have reported the mitochondrial effects of DXR while investigating cardiac toxicity induced by the treatment, mostly in pediatric cases. Here, we demonstrate that DXR alters the mitochondrial membrane composition associated with bioenergetic impairment and cell death in human cancer cells. The remodeling of the mitochondrial membrane was explained by phosphatidylserine decarboxylase (PSD) inhibition by DXR. PSD catalyzes phosphatidylethanolamine (PE) synthesis from phosphatidylserine (PS), and DXR altered the PS/PE ratio in the mitochondrial membrane. Moreover, we observed that DXR localized to the mitochondrial compartment and drug uptake was rapid. Evaluation of other topoisomerase II inhibitors did not show any impact on the mitochondrial membrane composition, indicating that the DXR effect was specific. Therefore, our findings revealed a side molecular target for DXR and PSD, potentially involved in DXR anti-cancer properties and the associated toxicity.

The multistress-induced Translocator protein (TSPO) differentially modulates storage lipids metabolism in seeds and seedlings.

Translocator proteins (TSPO) are conserved membrane proteins extensively studied in mammals, but their function is still unclear. Angiosperm TSPO are transiently induced by abiotic stresses in vegetative tissues. We showed previously that constitutive expression of the Arabidopsis TSPO (AtTSPO) could be detrimental to the cell. Degradation of AtTSPO requires an active autophagy pathway. We show here that genetic modifications of TSPO expression in plant and yeast cells reduce the levels of cytoplasmic lipid droplets (LD). Transgenic Arabidopsis seedlings overexpressing AtTSPO contain less LD as compared with wild type (WT). LD levels were increased in Arabidopsis AtTSPO knockout (KO) seedlings. Deletion of the Schizosaccharomyces pombe TSPO resulted in an increase in LD level in the cell. As compared with the WT, the mutant strain was more sensitive to cerulenin, an inhibitor of fatty acids and sterol biosynthesis. We found that in contrast with seedlings, overexpression of AtTSPO (OE) resulted in an up to 50% increase in seeds fatty acids as compared with WT. A time course experiment revealed that after 4 days of seed imbibition, the levels of triacylglycerol (TAG) was still higher in the OE seeds as compared with WT or KO seeds. However, the de novo synthesis of phospholipids and TAG after 24 h of imbibition was substantially reduced in OE seeds as compared with WT or KO seeds. Our findings support a plant TSPO role in energy homeostasis in a tissue-specific manner, enhancing fatty acids and LD accumulation in mature seeds and limiting LD levels in seedlings.

The importance of cardiolipin synthase for mitochondrial ultrastructure, respiratory function, plant development, and stress responses in Arabidopsis.

Cardiolipin (CL) is the signature phospholipid of the mitochondrial inner membrane. In animals and yeast (Saccharomyces cerevisiae), CL depletion affects the stability of respiratory supercomplexes and is thus crucial to the energy metabolism of obligate aerobes. In eukaryotes, the last step of CL synthesis is catalyzed by CARDIOLIPIN SYNTHASE (CLS), encoded by a single-copy gene. Here, we characterize a cls mutant in Arabidopsis thaliana, which is devoid of CL. In contrast to yeast cls, where development is little affected, Arabidopsis cls seedlings are slow developing under short-day conditions in vitro and die if they are transferred to long-day (LD) conditions. However, when transferred to soil under LD conditions under low light, cls plants can reach the flowering stage, but they are not fertile. The cls mitochondria display abnormal ultrastructure and reduced content of respiratory complex I/complex III supercomplexes. The marked accumulation of tricarboxylic acid cycle derivatives and amino acids demonstrates mitochondrial dysfunction. Mitochondrial and chloroplastic antioxidant transcripts are overexpressed in cls leaves, and cls protoplasts are more sensitive to programmed cell death effectors, UV light, and heat shock. Our results show that CLS is crucial for correct mitochondrial function and development in Arabidopsis under both optimal and stress conditions.

The Qb-SNARE Memb11 interacts specifically with Arf1 in the Golgi apparatus of Arabidopsis thaliana.

The SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins are critical for the function of the secretory pathway. The SNARE Memb11 is involved in membrane trafficking at the ER-Golgi interface. The aim of the work was to decipher molecular mechanisms acting in Memb11-mediated ER-Golgi traffic. In mammalian cells, the orthologue of Memb11 (membrin) is potentially involved in the recruitment of the GTPase Arf1 at the Golgi membrane. However molecular mechanisms associated to Memb11 remain unknown in plants. Memb11 was detected mainly at the cis-Golgi and co-immunoprecipitated with Arf1, suggesting that Arf1 may interact with Memb11. This interaction of Memb11 with Arf1 at the Golgi was confirmed by in vivo BiFC (Bimolecular Fluorescence Complementation) experiments. This interaction was found to be specific to Memb11 as compared to either Memb12 or Sec22. Using a structural bioinformatic approach, several sequences in the N-ter part of Memb11 were hypothesized to be critical for this interaction and were tested by BiFC on corresponding mutants. Finally, by using both in vitro and in vivo approaches, we determined that only the GDP-bound form of Arf1 interacts with Memb11. Together, our results indicate that Memb11 interacts with the GDP-bound form of Arf1 in the Golgi apparatus.

Cell wall constrains lateral diffusion of plant plasma-membrane proteins.

A cell membrane can be considered a liquid-phase plane in which lipids and proteins theoretically are free to diffuse. Numerous reports, however, describe retarded diffusion of membrane proteins in animal cells. This anomalous diffusion results from a combination of structuring factors including protein-protein interactions, cytoskeleton corralling, and lipid organization into microdomains. In plant cells, plasma-membrane (PM) proteins have been described as relatively immobile, but the control mechanisms that structure the PM have not been studied. Here, we use fluorescence recovery after photobleaching to estimate mobility of a set of minimal PM proteins. These proteins consist only of a PM-anchoring domain fused to a fluorescent protein, but their mobilities remained limited, as is the case for many full-length proteins. Neither the cytoskeleton nor membrane microdomain structure was involved in constraining the diffusion of these proteins. The cell wall, however, was shown to have a crucial role in immobilizing PM proteins. In addition, by single-molecule fluorescence imaging we confirmed that the pattern of cellulose deposition in the cell wall affects the trajectory and speed of PM protein diffusion. Regulation of PM protein dynamics by the plant cell wall can be interpreted as a mechanism for regulating protein interactions in processes such as trafficking and signal transduction.

ER Membrane Lipid Composition and Metabolism: Lipidomic Analysis.

Plant ER membranes are the major site of biosynthesis of several lipid families (phospholipids, sphingolipids, neutral lipids such as sterols and triacylglycerols). The structural diversity of lipids presents considerable challenges to comprehensive lipid analysis. This chapter will briefly review the various biosynthetic pathways and will detail several aspects of the lipid analysis: lipid extraction, handling, separation, detection, identification, and data presentation. The different tools/approaches used for lipid analysis will also be discussed in relation to the studies to be carried out on lipid metabolism and function.

The receptor kinase FERONIA regulates phosphatidylserine localization at the cell surface to modulate ROP signaling.

Cells maintain a constant dialog between the extracellular matrix and their plasma membrane to fine tune signal transduction processes. We found that the receptor kinase FERONIA (FER), which is a proposed cell wall sensor, modulates phosphatidylserine plasma membrane accumulation and nano-organization, a key regulator of Rho GTPase signaling in Arabidopsis. We demonstrate that FER is required for both Rho-of-Plant 6 (ROP6) nano-partitioning at the membrane and downstream production of reactive oxygen species upon hyperosmotic stimulus. Genetic and pharmacological rescue experiments indicate that phosphatidylserine is required for a subset of, but not all, FER functions. Furthermore, application of FER ligand shows that its signaling controls both phosphatidylserine membrane localization and nanodomains formation, which, in turn, tunes ROP6 signaling. Together, we propose that a cell wall-sensing pathway controls via the regulation of membrane phospholipid content, the nano-organization of the plasma membrane, which is an essential cell acclimation to environmental perturbations.

Glucosylceramide biosynthesis is involved in Golgi morphology and protein secretion in plant cells.

Lipids have an established role as structural components of membranes or as signalling molecules, but their role as molecular actors in protein secretion is less clear. The complex sphingolipid glucosylceramide (GlcCer) is enriched in the plasma membrane and lipid microdomains of plant cells, but compared to animal and yeast cells, little is known about the role of GlcCer in plant physiology. We have investigated the influence of GlcCer biosynthesis by glucosylceramide synthase (GCS) on the efficiency of protein transport through the plant secretory pathway and on the maintenance of normal Golgi structure. We determined that GlcCer is synthesized at the beginning of the plant secretory pathway [mainly endoplasmic reticulum (ER)] and that D,L-threo-1-phenyl-2-decanoyl amino-3-morpholino-propanol (PDMP) is a potent inhibitor of plant GCS activity in vitro and in vivo. By an in vivo confocal microscopy approach in tobacco leaves infiltrated with PDMP, we showed that the decrease in GlcCer biosynthesis disturbed the transport of soluble and membrane secretory proteins to the cell surface, as these proteins were partly retained intracellularly in the ER and/or Golgi. Electron microscopic observations of Arabidopsis thaliana root cells after high-pressure freezing and freeze substitution evidenced strong morphological changes in the Golgi bodies, pointing to a link between decreased protein secretion and perturbations of Golgi structure following inhibition of GlcCer biosynthesis in plant cells.

Sterols and Sphingolipids as New Players in Cell Wall Building and Apical Growth of Nicotiana tabacum L. Pollen Tubes.

Pollen tubes are tip-growing cells that create safe routes to convey sperm cells to the embryo sac for double fertilization. Recent studies have purified and biochemically characterized detergent-insoluble membranes from tobacco pollen tubes. These microdomains, called lipid rafts, are rich in sterols and sphingolipids and are involved in cell polarization in organisms evolutionarily distant, such as fungi and mammals. The presence of actin in tobacco pollen tube detergent-insoluble membranes and the preferential distribution of these domains on the apical plasma membrane encouraged us to formulate the intriguing hypothesis that sterols and sphingolipids could be a "trait d'union" between actin dynamics and polarized secretion at the tip. To unravel the role of sterols and sphingolipids in tobacco pollen tube growth, we used squalestatin and myriocin, inhibitors of sterol and sphingolipid biosynthesis, respectively, to determine whether lipid modifications affect actin fringe morphology and dynamics, leading to changes in clear zone organization and cell wall deposition, thus suggesting a role played by these lipids in successful fertilization.

The enpp4 ectonucleotidase regulates kidney patterning signalling networks in Xenopus embryos.

The enpp ectonucleotidases regulate lipidic and purinergic signalling pathways by controlling the extracellular concentrations of purines and bioactive lipids. Although both pathways are key regulators of kidney physiology and linked to human renal pathologies, their roles during nephrogenesis remain poorly understood. We previously showed that the pronephros was a major site of enpp expression and now demonstrate an unsuspected role for the conserved vertebrate enpp4 protein during kidney formation in Xenopus. Enpp4 over-expression results in ectopic renal tissues and, on rare occasion, complete mini-duplication of the entire kidney. Enpp4 is required and sufficient for pronephric markers expression and regulates the expression of RA, Notch and Wnt pathway members. Enpp4 is a membrane protein that binds, without hydrolyzing, phosphatidylserine and its effects are mediated by the receptor s1pr5, although not via the generation of S1P. Finally, we propose a novel and non-catalytic mechanism by which lipidic signalling regulates nephrogenesis.

A novel di-acidic motif facilitates ER export of the syntaxin SYP31.

It is generally accepted that ER protein export is largely influenced by the transmembrane domain (TMD). The situation is unclear for membrane-anchored proteins such as SNAREs, which are anchored to the membrane by their TMD at the C-terminus. For example, in plants, Sec22 and SYP31 (a yeast Sed5 homologue) have a 17 aa TMD but different locations (ER/Golgi and Golgi), indicating that TMD length alone is not sufficient to explain their targeting. To establish the identity of factors that influence SNARE targeting, mutagenesis and live cell imaging experiments were performed on SYP31. It was found that deletion of the entire N-terminus domain of SYP31 blocked the protein in the ER. Several deletion mutants of different parts of this N-terminus domain indicated that a region between the SNARE helices Hb and Hc is required for Golgi targeting. In this region, replacement of the aa sequence MELAD by GAGAG or MALAG retained the protein in the ER, suggesting that MELAD may function as a di-acidic ER export motif EXXD. This suggestion was further verified by replacing the established di-acidic ER export motif DLE of a type II Golgi protein AtCASP and a membrane-anchored type I chimaera, TMcCCASP, by MELAD or GAGAG. The MELAD motif allowed the proteins to reach the Golgi, whereas the motif GAGAG was found to be insufficient to facilitate ER protein export. Our analyses indicate that we have identified a novel and transplantable di-acidic motif that facilitates ER export of SYP31 and may function for type I and type II proteins in plants.

Developmental control of plant Rho GTPase nano-organization by the lipid phosphatidylserine.

Rho guanosine triphosphatases (GTPases) are master regulators of cell signaling, but how they are regulated depending on the cellular context is unclear. We found that the phospholipid phosphatidylserine acts as a developmentally controlled lipid rheostat that tunes Rho GTPase signaling in Arabidopsis Live superresolution single-molecule imaging revealed that the protein Rho of Plants 6 (ROP6) is stabilized by phosphatidylserine into plasma membrane nanodomains, which are required for auxin signaling. Our experiments also revealed that the plasma membrane phosphatidylserine content varies during plant root development and that the level of phosphatidylserine modulates the quantity of ROP6 nanoclusters induced by auxin and hence downstream signaling, including regulation of endocytosis and gravitropism. Our work shows that variations in phosphatidylserine levels are a physiological process that may be leveraged to regulate small GTPase signaling during development.

ER Membrane Lipid Composition and Metabolism: Lipidomic Analysis.

Plant ER membranes are the major site of biosynthesis of several lipid families (phospholipids, sphingolipids, neutral lipids such as sterols and triacylglycerols). The structural diversity of lipids presents considerable challenges to comprehensive lipid analysis. This chapter will briefly review the various biosynthetic pathways and will detail several aspects of the lipid analysis: lipid extraction, handling, separation, detection, identification, and data presentation. The different tools/approaches used for lipid analysis will also be discussed in relation to the studies to be carried out on lipid metabolism and function.

The odd one out: Arabidopsis reticulon 20 does not bend ER membranes but has a role in lipid regulation.

Reticulons are integral ER membrane proteins characterised by a reticulon homology domain comprising four transmembrane domains which results in the proteins sitting in the membrane in a W-topology. Here we report on a novel subgroup of reticulons with an extended N-terminal domain and in particular on arabidopsis reticulon 20. Using high resolution confocal microscopy we show that reticulon 20 is located in a unique punctate pattern on the ER membrane. Its closest homologue reticulon 19 labels the whole ER. Other than demonstrated for the other members of the reticulon protein family RTN20 and 19 do not display ER constriction phenotypes on over expression. We show that mutants in RTN20 or RTN19, respectively, display a significant change in sterol composition in roots indicating a role in lipid regulation. A third homologue in this family -3BETAHSD/D1- is unexpectedly localised to ER exit sites resulting in an intriguing location difference for the three proteins.

Constraint-Based Modeling Highlights Cell Energy, Redox Status and α-Ketoglutarate Availability as Metabolic Drivers for Anthocyanin Accumulation in Grape Cells Under Nitrogen Limitation.

Anthocyanin biosynthesis is regulated by environmental factors (such as light, temperature, and water availability) and nutrient status (such as carbon, nitrogen, and phosphate nutrition). Previous reports show that low nitrogen availability strongly enhances anthocyanin accumulation in non carbon-limited plant organs or cell suspensions. It has been hypothesized that high carbon-to-nitrogen ratio would lead to an energy excess in plant cells, and that an increase in flavonoid pathway metabolic fluxes would act as an "energy escape valve," helping plant cells to cope with energy and carbon excess. However, this hypothesis has never been tested directly. To this end, we used the grapevine Vitis vinifera L. cultivar Gamay Teinturier (syn. Gamay Freaux or Freaux Tintorier, VIVC #4382) cell suspension line as a model system to study the regulation of anthocyanin accumulation in response to nitrogen supply. The cells were sub-cultured in the presence of either control (25 mM) or low (5 mM) nitrate concentration. Targeted metabolomics and enzyme activity determinations were used to parametrize a constraint-based model describing both the central carbon and nitrogen metabolisms and the flavonoid (phenylpropanoid) pathway connected by the energy (ATP) and reducing power equivalents (NADPH and NADH) cofactors. The flux analysis (2 flux maps generated, for control and low nitrogen in culture medium) clearly showed that in low nitrogen-fed cells all the metabolic fluxes of central metabolism were decreased, whereas fluxes that consume energy and reducing power, were either increased (upper part of glycolysis, shikimate, and flavonoid pathway) or maintained (pentose phosphate pathway). Also, fluxes of flavanone 3ß-hydroxylase, flavonol synthase, and anthocyanidin synthase were strongly increased, advocating for a regulation of the flavonoid pathway by alpha-ketoglutarate levels. These results strongly support the hypothesis of anthocyanin biosynthesis acting as an energy escape valve in plant cells, and they open new possibilities to manipulate flavonoid production in plant cells. They do not, however, support a role of anthocyanins as an effective mechanism for coping with carbon excess in high carbon to nitrogen ratio situations in grape cells. Instead, constraint-based modeling output and biomass analysis indicate that carbon excess is dealt with by vacuolar storage of soluble sugars.

A Combinatorial Lipid Code Shapes the Electrostatic Landscape of Plant Endomembranes.

Membrane surface charge is critical for the transient, yet specific recruitment of proteins with polybasic regions to certain organelles. In eukaryotes, the plasma membrane (PM) is the most electronegative compartment of the cell, which specifies its identity. As such, membrane electrostatics is a central parameter in signaling, intracellular trafficking, and polarity. Here, we explore which are the lipids that control membrane electrostatics using plants as a model. We show that phosphatidylinositol-4-phosphate (PI4P), phosphatidic acidic (PA), and phosphatidylserine (PS) are separately required to generate the electrostatic signature of the plant PM. In addition, we reveal the existence of an electrostatic territory that is organized as a gradient along the endocytic pathway and is controlled by PS/PI4P combination. Altogether, we propose that combinatorial lipid composition of the cytosolic leaflet of organelles not only defines the electrostatic territory but also distinguishes different functional compartments within this territory by specifying their varying surface charges.

Immunolocalization and high affinity interactions of acyl-CoAs with proteins: an original study with anti-acyl-CoA antibodies.

Anti-acyl-Coenzyme A (acyl-CoA) antibodies were used to detect fatty acyl-CoAs in cultured rat hippocampal neurons, in which important lipid metabolism and transport occur. Hippocampus was chosen because of his involvement in many cerebral functions and diseases. Immunofluorescence experiments showed an intense labelling within neurites and cell bodies. Labelling seems to be associated with vesicles and membrane domains. We have shown by immunoblot experiments that the labelling corresponded to acyl-CoAs which were in strong interaction with proteins, without being covalently bound to them. Immunoprecipitation experiments, followed by proteomic analysis, showed that anti-acyl-CoA antibodies were also able to immunoprecipitate multiprotein complexes, principally related to vesicle trafficking and/or to membrane rafts.

Immunolocalization of long-chain acyl-CoAs in plant cells.

Long chain acyl-Coenzyme A esters (acyl-CoAs) are key substrates in many enzymic reactions of lipid metabolism. Due to their amphiphilic nature, the membrane localization of these molecules cannot be established by subcellular membrane fractionation and usual biochemical studies. We have developed another approach based on ultrastructural immunogold cytochemistry. To preserve the acyl-CoA membrane content, the plant material was freeze substituted and cryoembedded after short aldehyde fixation followed by quick freezing. Using Arabidopsis thaliana root cells and specific antibodies raised against acyl-CoAs, we show that acyl-CoAs are mainly localized in endoplasmic reticulum membranes. Our results demonstrate the value of cryo-methods for the accurate localization of labile metabolites in plant cells.