Role of Sphingosine Kinase 1 in Glucolipotoxicity-Induced Early Activation of Autophagy in INS-1 Pancreatic ß Cells.

Insulin-producing pancreatic ß cells play a crucial role in the regulation of glucose homeostasis, and their failure is a key event for diabetes development. Prolonged exposure to palmitate in the presence of elevated glucose levels, termed gluco-lipotoxicity, is known to induce ß cell apoptosis. Autophagy has been proposed to be regulated by gluco-lipotoxicity in order to favor ß cell survival. However, the role of palmitate metabolism in gluco-lipotoxcity-induced autophagy is presently unknown. We therefore treated INS-1 cells for 6 and 24 h with palmitate in the presence of low and high glucose concentrations and then monitored autophagy. Gluco-lipotoxicity induces accumulation of LC3-II levels in INS-1 at 6 h which returns to basal levels at 24 h. Using the RFP-GFP-LC3 probe, gluco-lipotoxicity increased both autophagosomes and autolysosmes structures, reflecting early stimulation of an autophagy flux. Triacsin C, a potent inhibitor of the long fatty acid acetyl-coA synthase, completely prevents LC3-II formation and recruitment to autophagosomes, suggesting that autophagic response requires palmitate metabolism. In contrast, etomoxir and bromo-palmitate, inhibitors of fatty acid mitochondrial ß-oxidation, are unable to prevent gluco-lipotoxicity-induced LC3-II accumulation and recruitment to autophagosomes. Moreover, bromo-palmitate and etomoxir potentiate palmitate autophagic response. Even if gluco-lipotoxicity raised ceramide levels in INS-1 cells, ceramide synthase 4 overexpression does not potentiate LC3-II accumulation. Gluco-lipotoxicity also still stimulates an autophagic flux in the presence of an ER stress repressor. Finally, selective inhibition of sphingosine kinase 1 (SphK1) activity precludes gluco-lipotoxicity to induce LC3-II accumulation. Moreover, SphK1 overexpression potentiates autophagic flux induced by gluco-lipotxicity. Altogether, our results indicate that early activation of autophagy by gluco-lipotoxicity is mediated by SphK1, which plays a protective role in ß cells.

Iron status and root cell morphology of Arabidopsis thaliana as modified by a bacterial ferri-siderophore.

We previously provided evidence for the contribution of pyoverdine to the iron nutrition of Arabidopsis. In the present article, we further analyze the mechanisms and physiology of the adaptations underlying plant iron nutrition through Fe(III)-pyoverdine (Fe(III)-pvd). An integrated approach combining microscopy and nanoscale secondary ion mass spectrometry (NanoSIMS) on plant samples was adopted to localize pyoverdine in planta and assess the impact of this siderophore on the plant iron status and root cellular morphology. The results support a possible plant uptake mechanism of the Fe(III)-pvd complex by epidermal root cells via a non-reductive process associated with the presence of more vesicles. Pyoverdine was transported to the central cylinder via the symplastic and/or trans-cellular pathway(s), suggesting a possible root-to-shoot translocation. All these processes led to enhanced plant iron nutrition, as previously shown. Overall, these findings suggest that bacterial siderophores contribute to plant iron uptake and homeostasis.

Four hundred years of cork imaging: New advances in the characterization of the cork structure.

In 1665, Robert Hooke was the first to observe cork cells and their characteristic hexagonal shape, using the first optical microscope, which was invented by him at that time. With the evolution of imaging techniques, the structure of cork has been analysed with greater accuracy over time. This work presents the latest advances in the characterization of this unique material through a multiscale approach. Such investigation brings new insight into the architecture of cork, particularly the differences between the cells of the phellem and those bordering the lenticels. In the latter case, cell differentiation from the lenticular phellogen was restricted to one cell layer, which leads to a cell wall that is 10 times thicker for lenticels. They also displayed a different chemical composition because of unsuberization and a high lignin content in lenticels. Such advances in the knowledge of the structure and composition of cork cells contributes to a better understanding of the macroporosity of cork, down to the nanoscale.

Mechanisms of astringency: Structural alteration of the oral mucosal pellicle by dietary tannins and protective effect of bPRPs.

The interaction of tannins with salivary proteins is involved in astringency. This paper focussed on saliva lining oral mucosae, the mucosal pellicle. Using a cell-based model, the impact of two dietary tannins (EgC and EgCG) on the mucosal pellicle structure and properties was investigated by microscopic techniques. The role of basic Proline-Rich-Proteins (bPRPs) in protecting the mucosal pellicle was also evaluated. At low (0.05 mM) tannin concentration, below the sensory detection threshold, the distribution of salivary mucins MUC5B on cells remained unaffected. At 0.5 and 1 mM, MUC5B-tannin aggregates were observed and their size increased with tannin concentration and with galloylation. In addition, 3 mM EgCG resulted in higher friction forces measured by AFM. In presence of bPRPs, the size distribution of aggregates was greatly modified and tended to resemble that of the "no tannin" condition, highlighting that bPRPs have a protective effect against the structural alteration induced by dietary tannins.

Changes in plastid proteome and structure in arbuscular mycorrhizal roots display a nutrient starvation signature.

During arbuscular mycorrhizal symbiosis, arbuscule-containing root cortex cells display a proliferation of plastids, a feature usually ascribed to an increased plant anabolism despite the lack of studies focusing on purified root plastids. In this study, we investigated mycorrhiza-induced changes in plastidic pathways by performing a label-free comparative subcellular quantitative proteomic analysis targeted on plastid-enriched fractions isolated from Medicago truncatula roots, coupled to a cytological analysis of plastid structure. We identified 490 root plastid protein candidates, among which 79 changed in abundance upon mycorrhization, as inferred from spectral counting. According to cross-species sequence homology searches, the mycorrhiza-responsive proteome was enriched in proteins experimentally localized in thylakoids, whereas it was depleted of proteins ascribed predominantly to amyloplasts. Consistently, the analysis of plastid morphology using transmission electron microscopy indicated that starch depletion associated with the proliferation of membrane-free and tubular membrane-containing plastids was a feature specific to arbusculated cells. The loss of enzymes involved in carbon/nitrogen assimilation and provision of reducing power, coupled to macromolecule degradation events in the plastid-enriched fraction of mycorrhizal roots that paralleled lack of starch accumulation in arbusculated cells, lead us to propose that arbuscule functioning elicits a nutrient starvation and an oxidative stress signature that may prime arbuscule breakdown.

Revisiting Plant Plasma Membrane Lipids in Tobacco: A Focus on Sphingolipids.

The lipid composition of plasma membrane (PM) and the corresponding detergent-insoluble membrane (DIM) fraction were analyzed with a specific focus on highly polar sphingolipids, so-called glycosyl inositol phosphorylceramides (GIPCs). Using tobacco (Nicotiana tabacum) 'Bright Yellow 2' cell suspension and leaves, evidence is provided that GIPCs represent up to 40 mol % of the PM lipids. Comparative analysis of DIMs with the PM showed an enrichment of 2-hydroxylated very-long-chain fatty acid-containing GIPCs and polyglycosylated GIPCs in the DIMs. Purified antibodies raised against these GIPCs were further used for immunogold-electron microscopy strategy, revealing the distribution of polyglycosylated GIPCs in domains of 35 ± 7 nm in the plane of the PM. Biophysical studies also showed strong interactions between GIPCs and sterols and suggested a role for very-long-chain fatty acids in the interdigitation between the two PM-composing monolayers. The ins and outs of lipid asymmetry, raft formation, and interdigitation in plant membrane biology are finally discussed.

The membrane-associated MUC1 improves adhesion of salivary MUC5B on buccal cells. Application to development of an in vitro cellular model of oral epithelium.

OBJECTIVES: The mucosal pellicle is a thin layer of salivary proteins, mostly MUC5B mucins, anchored to epithelial oral cells. This pellicle is involved in protection of oral mucosae against abrasion, pathogenic microorganisms or chemical xenobiotics. The present study aimed at studying the involvement of MUC1 in mucosal pellicle formation and more specifically in salivary MUC5B binding using a cell-based model of oral epithelium. DESIGN: MUC1 mRNAs were not detected in TR146 cells, and therefore a stable cell line named TR146/MUC1 expressing this protein was developed by transfection. TR146 and TR146/MUC1 were incubated with human saliva in order to evaluate retention of MUC5B by epithelial cells. RESULTS: The cell surface of both TR146 and TR146/MUC1 was typical of a squamous non-keratinized epithelium, with the presence of numerous microplicae. After incubation for 2h with saliva diluted in culture medium (1:1) and two washes with PBS, saliva deposits on cells appeared as a loose filamentous thin network. MUC5B fluorescent immunostaining evidenced a heterogeneous lining of confluent cell cultures by this salivary mucin but with higher fluorescence on TR146/MUC1 cells. Semi-quantification of MUC5B bound to cells confirmed a better retention by TR146/MUC1, evaluated by Dot Blot (+34.1%, p<0.05) or by immunocytochemistry (+44%, p<0.001). CONCLUSION: The membrane-bound mucin MUC1 is a factor enhancing the formation of the mucosal pellicle by increasing the binding of salivary MUC5B to oral epithelial cells. An in vitro model suitable to study specifically the function and properties of the mucosal pellicle is proposed.

Nanovectorization of TRAIL with single wall carbon nanotubes enhances tumor cell killing.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL or Apo2L) is a member of the tumor necrosis factor (TNF) superfamily. This type II transmembrane protein is able to bound specifically to cancer cell receptors (i.e., TRAIL-R1 (or DR4) and TRAIL-R2 (or DR5)) and to induce apoptosis without being toxic for healthy cells. Because membrane-bound TRAIL induces stronger receptor aggregation and apoptosis than soluble TRAIL, we proposed here to vectorize TRAIL using single-walled carbon nanotubes (SWCNTs) to mimic membrane TRAIL. Owing to their exceptional and revolutional properties, carbon nanotubes, especially SWCNTs, are used in a wide range of physical or, now, medical applications. Indeed due to their high mechanical resistance, their high flexibility and their hydrophobicity, SWCNTs are known to rapidly diffuse in an aqueous medium such as blood, opening the way of development of new drug nanovectors (or nanocarriers). Our TRAIL-based SWCNTs nanovectors proved to be more efficient than TRAIL alone death receptors in triggering cancer cell killing. These NPTs increased TRAIL pro-apoptotic potential by nearly 20-fold in different Human tumor cell lines including colorectal, nonsmall cell lung cancer, or hepatocarcinomas. We provide thus a proof-of-concept that TRAIL nanovector derivatives based on SWCNT may be useful to future nanomedicine therapies.

Long-chain bases and their phosphorylated derivatives differentially regulate cryptogein-induced production of reactive oxygen species in tobacco (Nicotiana tabacum) BY-2 cells.

The proteinaceous elicitor cryptogein triggers defence reactions in Nicotiana tabacum (tobacco) through a signalling cascade, including the early production of reactive oxygen species (ROS) by the plasma membrane (PM)-located tobacco respiratory burst oxidase homologue D (NtRbohD). Sphingolipid long-chain bases (LCBs) are emerging as potent positive regulators of plant defence-related mechanisms. This led us to question whether both LCBs and their phosphorylated derivatives (LCB-Ps) are involved in the early signalling process triggered by cryptogein in tobacco BY-2 cells. Here, we showed that cryptogein-induced ROS production was inhibited by LCB kinase (LCBK) inhibitors. Additionally, Arabidopsis thaliana sphingosine kinase 1 and exogenously supplied LCB-Ps increased cryptogein-induced ROS production, whereas exogenously supplied LCBs had a strong opposite effect, which was not driven by a reduction in cellular viability. Immunogold-electron microscopy assay also revealed that LCB-Ps are present in the PM, which fits well with the presence of a high LCBK activity associated with this fraction. Our data demonstrate that LCBs and LCB-Ps differentially regulate cryptogein-induced ROS production in tobacco BY-2 cells, and support a model in which a cooperative synergism between LCBK/LCB-Ps and NtRbohD/ROS in the cryptogein signalling pathway is likely at the PM in tobacco BY-2 cells.

Dynamic changes in the subcellular distribution of the tobacco ROS-producing enzyme RBOHD in response to the oomycete elicitor cryptogein.

Plant NADPH oxidases, also known as respiratory burst oxidase homologues (RBOHs), have been identified as a major source of reactive oxygen species (ROS) during plant-microbe interactions. The subcellular localization of the tobacco (Nicotiana tabacum) ROS-producing enzyme RBOHD was examined in Bright Yellow-2 cells before and after elicitation with the oomycete protein cryptogein using electron and confocal microscopy. The plasma membrane (PM) localization of RBOHD was confirmed and immuno-electron microscopy on purified PM vesicles revealed its distribution in clusters. The presence of the protein fused to GFP was also seen in intracellular compartments, mainly Golgi cisternae. Cryptogein induced, within 1h, a 1.5-fold increase in RBOHD abundance at the PM and a concomitant decrease in the internal compartments. Use of cycloheximide revealed that most of the proteins targeted to the PM upon elicitation were not newly synthesized but may originate from the Golgi pool. ROS accumulation preceded RBOHD transcript- and protein-upregulation, indicating that ROS resulted from the activation of a PM-resident pool of enzymes, and that enzymes newly addressed to the PM were inactive. Taken together, the results indicate that control of RBOH abundance and subcellular localization may play a fundamental role in the mechanism of ROS production.

Immunocytological detection of salivary mucins (MUC5B) on the mucosal pellicle lining human epithelial buccal cells.

The mucosal pellicle is defined as the protein film adsorbed onto oral mucosa. This study aimed at characterizing the ultrastructure of human epithelial buccal cells and localizing salivary mucins MUC5B, a major constituent of the mucosal pellicle. Cells were sampled from the buccal surface and prepared for Transmission Electron Microscopy using high-pressure freezing/cryosubstitution followed by immunogold labelling of MUC5B. Morphologically, cells were visualized as typical cells of the superficial layer of a squamous nonkeratinized epithelium with a partly degraded plasma membrane. The outer surface of the plasma membrane was lined with a biological material of medium electron density. MUC5B were detected in the extracellular space, and particularly in the vicinity of the plasma membrane, sometimes onto fibrils protruding from the membrane. This area was, therefore, considered as constituting the mucosal pellicle, which appeared as a mixed film of both salivary and epithelial components. The distribution of gold particles suggested that the surface of the pellicle was not uniform, and that the film thickness could reach up to 100 nm. This work showed the feasibility of visualizing and characterizing the mucosal pellicle directly on human epithelial buccal cells sampled in a noninvasive manner.

Membrane rafts in plant cells.

Over the past five years, the structure, composition and possible functions of membrane raft-like domains on plant plasma membranes (PM) have been described. Proteomic analyses have indicated that a high proportion of proteins associated with detergent-insoluble membranes (DIMs), supposed to contain raft-like domains isolated from the PM, might be involved in signalling pathways. Recently, the dynamic association of specific proteins with the DIM fraction upon environmental stress has been reported. Innovative imaging methods have shown that lateral segregation of lipids and proteins exists at the nanoscale level in the plant PM, correlating detergent insolubility and membrane-domain localization of presumptive raft proteins. These data suggest a role for plant rafts as signal transduction platforms, similar to those documented for mammalian cells.

Plasma membrane sterol complexation, generated by filipin, triggers signaling responses in tobacco cells.

The effects of changes in plasma membrane (PM) sterol lateral organization and availability on the control of signaling pathways have been reported in various animal systems, but rarely assessed in plant cells. In the present study, the pentaene macrolide antibiotic filipin III, commonly used in animal systems as a sterol sequestrating agent, was applied to tobacco cells. We show that filipin can be used at a non-lethal concentration that still allows an homogeneous labeling of the plasma membrane and the formation of filipin-sterol complexes at the ultrastructural level. This filipin concentration triggers a rapid and transient NADPH oxidase-dependent production of reactive oxygen species, together with an increase in both medium alkalinization and conductivity. Pharmacological inhibition studies suggest that these signaling events may be regulated by phosphorylations and free calcium. By conducting FRAP experiments using the di-4-ANEPPDHQ probe and spectrofluorimetry using the Laurdan probe, we provide evidence for a filipin-induced increase in PM viscosity that is also regulated by phosphorylations. We conclude that filipin triggers ligand-independent signaling responses in plant cells. The present findings strongly suggest that changes in PM sterol availability could act as a sensor of the modifications of cell environment in plants leading to adaptive cell responses through regulated signaling processes.

Lateral reorganization of plasma membrane is involved in the yeast resistance to severe dehydration.

In this study, we investigated the kinetic and the magnitude of dehydrations on yeast plasma membrane (PM) modifications because this parameter is crucial to cell survival. Functional (permeability) and structural (morphology, ultrastructure, and distribution of the protein Sur7-GFP contained in sterol-rich membrane microdomains) PM modifications were investigated by confocal and electron microscopy after progressive (non-lethal) and rapid (lethal) hyperosmotic perturbations. Rapid cell dehydration induced the formation of many PM invaginations followed by membrane internalization of low sterol content PM regions with time. Permeabilization of the plasma membrane occurred during the rehydration stage because of inadequacies in the membrane surface and led to cell death. Progressive dehydration conducted to the formation of some big PM pleats without membrane internalization. It also led to the modification of the distribution of the Sur7-GFP microdomains, suggesting that a lateral rearrangement of membrane components occurred. This event is a function of time and is involved in the particular deformations of the PM during a progressive perturbation. The maintenance of the repartition of the microdomains during rapid perturbations consolidates this assumption. These findings highlight that the perturbation kinetic influences the evolution of the PM organization and indicate the crucial role of PM lateral reorganization in cell survival to hydric perturbations.

Vector transmission of a plant-pathogenic bacterium in the Arsenophonus clade sharing ecological traits with facultative insect endosymbionts.

The planthopper Pentastiridius leporinus (Hemiptera: Cixiidae) is the major vector of a nonculturable plant-pathogenic gamma-3 proteobacterium associated with a disease of sugar beet called syndrome "basses richesses" (SBR). The bacterium, here called SBR bacterium, belongs to the Arsenophonous clade, which includes mostly insect-associated facultative symbionts. Assays using field-collected planthopper nymphs and adults were carried out to investigate the interaction of SBR bacterium with the insect vector and its transmission to sugar beet. Field-collected planthoppers showed a percentage of infection that averaged from 57% for early instar nymphs to near 100% for late instar nymphs and emerging adults. SBR bacterium was persistently transmitted by emerging adults. Root-feeding nymphs were able to inoculate SBR bacterium to sugar beet. The bacterium was transmitted vertically from infected parental females to their respective offspring with an average frequency of 30%. Real-time polymerase chain reaction assays on dissected planthopper internal organs revealed a high concentration of the bacterium within male and female reproductive organs and within female salivary glands. SBR-like bacteria were observed through transmission electron microscopy in the cytoplasm of different insect organs including ovaries, salivary glands, and guts with no evidence for cytological disorders. SBR bacterium seems to share common ecological traits of insect-transmitted plant pathogens and facultative insect endosymbionts suggesting it may have evolved primarily as an insect-associated bacterium.

NADPH oxidase-mediated reactive oxygen species production: subcellular localization and reassessment of its role in plant defense.

Chemiluminescence detection of reactive oxygen species (ROS) triggered in tobacco BY-2 cells by the fungal elicitor cryptogein was previously demonstrated to be abolished in cells transformed with an antisense construct of the plasma membrane NADPH oxidase, NtrbohD. Here, using electron microscopy, it has been confirmed that the first hydrogen peroxide production occurring a few minutes after challenge of tobacco cells with cryptogein is plasma membrane located and NtrbohD mediated. Furthermore, the presence of NtrbohD in detergent-resistant membrane fractions could be associated with the presence of NtrbohD-mediated hydrogen peroxide patches along the plasma membrane. Comparison of the subcellular localization of ROS in wild-type tobacco and in plants transformed with antisense constructs of NtrbohD revealed that this enzyme is also responsible for the hydrogen peroxide production occurring at the plasma membrane after infiltration of tobacco leaves with cryptogein. Finally, the reactivity of wild-type and transformed plants to the elicitor and their resistance against the pathogenic oomycete Phytophthora parasitica were examined. NtrbohD-mediated hydrogen peroxide production does not seem determinant for either hypersensitive response development or the establishment of acquired resistance but it is most likely involved in the signaling pathways associated with the protection of the plant cell.

Remorin, a solanaceae protein resident in membrane rafts and plasmodesmata, impairs potato virus X movement.

Remorins (REMs) are proteins of unknown function specific to vascular plants. We have used imaging and biochemical approaches and in situ labeling to demonstrate that REM clusters at plasmodesmata and in approximately 70-nm membrane domains, similar to lipid rafts, in the cytosolic leaflet of the plasma membrane. From a manipulation of REM levels in transgenic tomato (Solanum lycopersicum) plants, we show that Potato virus X (PVX) movement is inversely related to REM accumulation. We show that REM can interact physically with the movement protein TRIPLE GENE BLOCK PROTEIN1 from PVX. Based on the localization of REM and its impact on virus macromolecular trafficking, we discuss the potential for lipid rafts to act as functional components in plasmodesmata and the plasma membrane.

The plant defense elicitor cryptogein stimulates clathrin-mediated endocytosis correlated with reactive oxygen species production in bright yellow-2 tobacco cells.

The plant defense elicitor cryptogein triggers well-known biochemical events of early signal transduction at the plasma membrane of tobacco (Nicotiana tabacum) cells, but microscopic observations of cell responses related to these early events were lacking. We determined that internalization of the lipophilic dye FM4-64, which is a marker of endocytosis, is stimulated a few minutes after addition of cryptogein to tobacco Bright Yellow-2 (BY-2) cells. This stimulation is specific to the signal transduction pathway elicited by cryptogein because a lipid transfer protein, which binds to the same receptor as cryptogein but without triggering signaling, does not increase endocytosis. To define the nature of the stimulated endocytosis, we quantified clathrin-coated pits (CCPs) forming on the plasma membrane of BY-2 cells. A transitory stimulation of this morphological event by cryptogein occurs within the first 15 min. In the presence of cryptogein, increases in both FM4-64 internalization and clathrin-mediated endocytosis are specifically blocked upon treatment with 5 microm tyrphostin A23, a receptor-mediated endocytosis inhibitor. The kinetics of the transient increase in CCPs at the plasma membrane coincides with that of transitory reactive oxygen species (ROS) production occurring within the first 15 min after elicitation. Moreover, in BY-2 cells expressing NtrbohD antisense cDNA, which are unable to produce ROS when treated with cryptogein, the CCP stimulation is inhibited. These results indicate that the very early endocytic process induced by cryptogein in tobacco is due, at least partly, to clathrin-mediated endocytosis and is dependent on ROS production by the NADPH oxidase NtrbohD.

Cytotoxic oxysterols induce caspase-independent myelin figure formation and caspase-dependent polar lipid accumulation.

Oxysterols, mainly those oxidized at the C7 position, induce a complex mode of cell death exhibiting some characteristics of apoptosis associated with a rapid induction of lipid rich multilamellar cytoplasmic structures (myelin figures) observed in various pathologies including atherosclerosis. The aim of this study was to determine the relationships between myelin figure formation, cell death, and lipid accumulation in various cell lines (U937, THP-1, MCF-7 [caspase-3 deficient], A7R5) treated either with oxysterols (7-ketocholesterol [7KC], 7beta-hydroxycholesterol, cholesterol-5alpha,6alpha-epoxide, cholesterol-5beta,6beta-epoxide, 25-hydroxycholesterol) or cytotoxic drugs (etoposide, daunorubicin, tunicamycin, rapamycin). Cell death was assessed by the measurement of cellular permeability with propidium iodide, characterization of the morphological aspect of the nuclei with Hoechst 33342, and identification of myelin figures by transmission electron microscopy. Nile Red staining (distinguishing neutral and polar lipids) was used to identify lipid content by flow cytometry and spectral imaging microscopy. Whatever the cells considered, myelin figures were only observed with cytotoxic oxysterols (7KC, 7beta-hydroxycholesterol, cholesterol-5beta, 6beta-epoxide), and their formation was not inhibited by the broad spectrum caspase inhibitor z-VAD-fmk. When U937 cells were treated with oxysterols or cytotoxic drugs, polar lipid accumulation was mainly observed with 7KC and 7beta-hydroxycholesterol. The highest polar lipid accumulation, which was triggered by 7KC, was counteracted by z-VAD-fmk. These findings demonstrate that myelin figure formation is a caspase-independent event closely linked with the cytotoxicity of oxysterols, and they highlight a relationship between caspase activity and polar lipid accumulation.