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.Eukaryotic plasma membranes (PMs) are composed of three main classes of lipids, glycerolipids, sphingolipids, and sterols, which may account for up to 100,000 different molecular species (Yetukuri et al., 2008; Shevchenko and Simons, 2010). Overall, all glycerolipids share the same molecular moieties in plants, animals, and fungi. By contrast, sterols and sphingolipids are different and specific to each kingdom. For instance, the plant PM contains an important number of sterols, among which β-sitosterol, stigmasterol, and campesterol predominate (Furt et al., 2011). In addition to free sterols, phytosterols can be conjugated to form steryl glycosides (SG) and acyl steryl glycosides (ASG) that represent up to approximately 15% of the tobacco (Nicotiana tabacum) PM (Furt et al., 2010). As for sphingolipids, sphingomyelin, the major phosphosphingolipid in animals, which harbors a phosphocholine as a polar head, is not detected in plants. Glycosyl inositol phosphorylceramides (GIPCs) are the major class of sphingolipids in plants, but they are absent in animals (Sperling and Heinz, 2003; Pata et al., 2010). Sphingolipidomic approaches identified up to 200 plant sphingolipids (for review, see Pata et al., 2010; Cacas et al., 2013).Although GIPCs belong to one of the earliest classes of plant sphingolipids that were identified in the late 1950s (Carter et al., 1958), only a few GIPCs have been structurally characterized to date because of their high polarity and a limited solubility in typical lipid extraction solvents. For these reasons, they were systematically omitted from published plant PM lipid composition. GIPCs are formed by the addition of an inositol phosphate to the ceramide moiety, the inositol headgroup of which can then undergo several glycosylation steps. The dominant glycan structure, composed of a hexose-GlcA linked to the inositol, is called series A. Polar heads containing three to seven sugars, so-called series B to F, have been identified and appeared to be species specific (Buré et al., 2011; Cacas et al., 2013; Mortimer et al., 2013). The ceramide moiety of GIPCs consists of a long-chain base (LCB), mainly t18:0 (called phytosphingosine) or t18:1 compounds (for review, see Pata et al., 2010), to which is amidified a very-long-chain fatty acid (VLCFA), the latter of which is mostly 2-hydroxylated (hVLCFA) with an odd or even number of carbon atoms. In plants, little is known about the subcellular localization of GIPCs. It is assumed, however, that they would be highly represented in the PM (Worrall et al., 2003; Sperling et al., 2005), even if this remains to be experimentally proven. The main argument supporting such an assumption is the strong enrichment of trihydroxylated LCB (t18:n) in detergent-insoluble membrane (DIM) fractions (Borner et al., 2005; Lefebvre et al., 2007), LCB being known to be predominant in GIPC’s core structure as aforementioned.In addition to this chemical complexity, lipids are not evenly distributed within the PM. Sphingolipids and sterols can preferentially interact with each other and segregate to form microdomains dubbed the membrane raft (Simons and Toomre, 2000). The membrane raft hypothesis suggests that lipids play a regulatory role in mediating protein clustering within the bilayer by undergoing phase separation into liquid-disordered and liquid-ordered phases. The liquid-ordered phase, termed the membrane raft, was described as enriched in sterol and saturated sphingolipids and is characterized by tight lipid packing. Proteins, which have differential affinities for each phase, may become enriched in, or excluded from, the liquid-ordered phase domains to optimize the rate of protein-protein interactions and maximize signaling processes. In animals, rafts have been implicated in a huge range of cellular processes, such as hormone signaling, membrane trafficking in polarized epithelial cells, T cell activation, cell migration, and the life cycle of influenza and human immunodeficiency viruses (Simons and Ikonen, 1997; Simons and Gerl, 2010). In plants, evidence is increasing that rafts are also involved in signal transduction processes and membrane trafficking (for review, see Mongrand et al., 2010; Simon-Plas et al., 2011; Cacas et al., 2012a).Moreover, lipids are not evenly distributed between the two leaflets of the PM. Within the PM of eukaryotic cells, sphingolipids are primarily located in the outer monolayer, whereas unsaturated phospholipids are predominantly exposed on the cytosolic leaflet. This asymmetrical distribution has been well established in human red blood cells, in which the outer leaflet contains sphingomyelin, phosphatidylcholine, and a variety of glycolipids like gangliosides. By contrast, the cytoplasmic leaflet is composed mostly of phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and their phosphorylated derivatives (Devaux and Morris, 2004). With regard to sphingolipids and glycerolipids, the asymmetry of the former is established during their biosynthesis and that of the latter requires ATPases such as the aminophospholipid translocase that transports lipids from the outer to the inner leaflet as well as multiple drug resistance proteins that transport phosphatidylcholine in the opposite direction (Devaux and Morris, 2004). This ubiquitous scheme encountered in animal cells could apply in plant cells as proposed (Tjellstrom et al., 2010). Indeed, the authors showed that there is a pronounced transverse lipid asymmetry in root at the PM. Phospholipids and galactolipids dominate the cytosolic leaflet, whereas the apoplastic leaflet is enriched in sphingolipids and sterols.From such a high diversity of the plant PM thus arises the question of the respective contribution of lipids to membrane suborganization. Our group recently tackled this aspect by characterizing the order level of liposomes prepared from various plant lipids and labeled with the environment-sensitive probe di-4-ANEPPDHQ (Grosjean et al., 2015). Fluorescence spectroscopy experiments showed that, among phytosterols, campesterol exhibits the strongest ability to order model membranes. In agreement with these data, spatial analysis of the membrane organization through multispectral confocal microscopy pointed to the strong ability of campesterol to promote liquid-ordered domain formation and organize their spatial distribution at the membrane surface. Conjugated sterols also exhibit a striking ability to order membranes. In addition, GIPCs enhance the sterol-induced ordering effect by emphasizing the formation and increasing the size of sterol-dependent ordered domains.The aim of this study was to reinvestigate the lipid composition and organization of the PM with a particular focus on GIPCs using tobacco leaves and cv Bright Yellow 2 (BY-2) cell cultures as models. Analyzing all membrane lipid classes at once, including sphingolipids, is challenging because they all display dramatically different chemical polarity, from very apolar (like free sterols) to highly polar (like polyglycosylated GIPCs) molecules. Most lipid extraction techniques published thus far use a chloroform/methanol mixture and phase partition to remove contaminants, resulting in the loss GIPCs, which remain in the aqueous phase, unextracted in the insoluble pellet, or at the interphase (Markham et al., 2006). In order to gain access to both glycerolipid and sphingolipid species at a glance, we developed a protocol whereby the esterifed or amidified fatty acids were hydrolyzed from the glycerol backbone (glycerolipids) or the LCB (sphingolipids) of membrane lipids, respectively. Fatty acids were then analyzed by gas chromatography-mass spectrometry (GC-MS) with appropriate internal standards for quantification. We further proposed that the use of methyl tert-butyl ether (MTBE) ensures the extraction of all classes of plant polar lipids. Our results indicate that GIPCs represent up to 40 mol % of total tobacco PM lipids. Interestingly, polyglycolyslated GIPCs are 5-fold enriched in DIMs of BY-2 cells when compared with the PM. Further investigation led us to develop a preparative purification procedure that allowed us to obtain enough material to raise antibodies against GIPCs. Using immunogold labeling on PM vesicles, it was found that polyglycosylated GIPCs cluster in membrane nanodomains, strengthening the idea that lateral nanosegregation of sphingolipids takes place at the PM in plants. Multispectral confocal microscopy was performed on vesicles prepared using GIPCs, phospholipids, and sterols and labeled with the environment-sensitive probe di-4-ANEPPDHQ. Our results show that, despite different fatty acid and polar head compositions, GIPCs extracted from tobacco leaves and BY-2 cells have a similar intrinsic propensity of enhancing vesicle global order together with sterols. Assuming that GIPCs are mostly present in the outer leaflet of the PM, interactions between sterols and sphingolipids were finally studied by the Langmuir monolayer technique, and the area of a single molecule of GIPC, or in interaction with phytosterols, was calculated. Using the calculation docking method, the energy of interaction between GIPCs and phytosterols was determined. A model was proposed in which GIPCs and phytosterols interact together to form liquid-ordered domains and in which the VLCFAs of GIPCs promote the interdigitation of the two membrane leaflets. The implications of domain formation and the asymmetrical distribution of lipids at the PM in plants are also discussed. Finally, we propose a model that reconsiders the intricate organization of the plant PM bilayer.     

Display of whole proteins on inner and outer surfaces of grapevine fanleaf virus‐like particles

Virus‐like particles (VLPs) derived from nonenveloped viruses result from the self‐assembly of capsid proteins (CPs). They generally show similar structural features to viral particles but are noninfectious and their inner cavity and outer surface can potentially be adapted to serve as nanocarriers of great biotechnological interest. While a VLP outer surface is generally amenable to chemical or genetic modifications, encaging a cargo within particles can be more complex and is often limited to small molecules or peptides. Examples where both inner cavity and outer surface have been used to simultaneously encapsulate and expose entire proteins remain scarce. Here, we describe the production of spherical VLPs exposing fluorescent proteins at either their outer surface or inner cavity as a result of the self‐assembly of a single genetically modified viral structural protein, the CP of grapevine fanleaf virus (GFLV). We found that the N‐ and C‐terminal ends of the GFLV CP allow the genetic fusion of proteins as large as 27 kDa and the plant‐based production of nucleic acid‐free VLPs. Remarkably, expression of N‐ or C‐terminal CP fusions resulted in the production of VLPs with recombinant proteins exposed to either the inner cavity or the outer surface, respectively, while coexpression of both fusion proteins led to the formation hybrid VLP, although rather inefficiently. Such properties are rather unique for a single viral structural protein and open new potential avenues for the design of safe and versatile nanocarriers, particularly for the targeted delivery of bioactive molecules.     

Bioaerosol biomonitoring: Sampling optimization for molecular microbial ecology

Bioaerosols (or biogenic aerosols) have largely been overlooked by molecular ecologists. However, this is rapidly changing as bioaerosols play key roles in public health, environmental chemistry and the dispersal ecology of microbes. Due to the low environmental concentrations of bioaerosols, collecting sufficient biomass for molecular methods is challenging. Currently, no standardized methods for bioaerosol collection for molecular ecology research exist. Each study requires a process of optimization, which greatly slows the advance of bioaerosol science. Here, we evaluated air filtration and liquid impingement for bioaerosol sampling across a range of environmental conditions. We also investigated the effect of sampling matrices, sample concentration strategies and sampling duration on DNA yield. Air filtration using polycarbonate filters gave the highest recovery, but due to the faster sampling rates possible with impingement, we recommend this method for fine ‐scale temporal/spatial ecological studies. To prevent bias for the recovery of Gram‐positive bacteria, we found that the matrix for impingement should be phosphate‐buffered saline. The optimal method for bioaerosol concentration from the liquid matrix was centrifugation. However, we also present a method using syringe filters for rapid in‐field recovery of bioaerosols from impingement samples, without compromising microbial diversity for high ‐throughput sequencing approaches. Finally, we provide a resource that enables molecular ecologists to select the most appropriate sampling strategy for their specific research question.     

The cold-inducible RNA-binding protein migrates from the nucleus to cytoplasmic stress granules by a methylation-dependent mechanism and acts as a translational repressor

The cold-inducible RNA-binding protein (CIRP) is a nuclear 18-kDa protein consisting of an amino-terminal RNA Recognition Motif (RRM) and a carboxyl-terminal domain containing several RGG motifs. First characterized for its overexpression upon cold shock, CIRP is also induced by stresses such as UV irradiation and hypoxia. Here, we investigated the expression as well as the subcellular localization of CIRP in response to other stress conditions. We demonstrate that oxidative stress leads to the migration of CIRP to stress granules (SGs) without alteration of expression. Stress granules are dynamic cytoplasmic foci at which stalled translation initiation complexes accumulate in cells subjected to environmental stress. Relocalization of CIRP into SGs also occurs upon other cytoplasmic stresses (osmotic pressure or heat shock) as well as in response to stresses of the endoplasmic reticulum. CIRP migration into SGs is independent from TIA-1 which has been previously reported to be a general mediator of SG formation, thereby suggesting the existence of multiple pathways leading to SG formation. Moreover, deletion mutants revealed that both RGG and RRM domains can independently promote CIRP migration into SGs. However, the methylation of arginine residues in the RGG domain is necessary for CIRP to exit the nucleus to be further recruited into SGs. By RNA-tethering experiments, we also show that CIRP down-regulates mRNA translation and that this activity is carried by the carboxyl-terminal RG-enriched domain. Altogether, our findings further reveal the diversity of mechanisms by which CIRP is regulated by environmental stresses and provide new insights into CIRP cytoplasmic function.     

Assembly of the <Emphasis Type="Italic">Tc1</Emphasis> and <Emphasis Type="Italic">mariner</Emphasis> transposition initiation complexes depends on the origins of their transposase DNA binding domains

In this review, we focus on the assembly of DNA/protein complexes that trigger transposition in eukaryotic members of the IS630–Tc1–mariner (ITm) super-family, the Tc1- and mariner-like elements (TLEs and MLEs). Elements belonging to this super-family encode transposases with DNA binding domains of different origins, and recent data indicate that the chimerization of functional domains has been an important evolutionary aspect in the generation of new transposons within the ITm super-family. These data also reveal that the inverted terminal repeats (ITRs) at the ends of transposons contain three kinds of motif within their sequences. The first two are well known and correspond to the cleavage site on the outer ITR extremities, and the transposase DNA binding site. The organization of ITRs and of the transposase DNA binding domains implies that differing pathways are used by MLEs and TLEs to regulate transposition initiation. These differences imply that the ways ITRs are recognized also differ leading to the formation of differently organized synaptic complexes. The third kind of motif is the transposition enhancers, which have been found in almost all the functional MLEs and TLEs analyzed to date. Finally, in vitro and in vivo assays of various elements all suggest that the transposition initiation complex is not formed randomly, but involves a mechanism of oriented transposon scanning. Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at . An erratum to this article can be found at     

Reversible hexacoordination of alpha-hemoglobin-stabilizing protein (AHSP)/alpha-hemoglobin Versus pressure. Evidence for protection of the alpha-chains by their chaperone

Using high hydrostatic pressure or hydrogen peroxide as perturbing agents, we demonstrate a protective effect of the chaperone AHSP for the alpha-chains of Hb. High pressure induces an irreversible aggregation of the ferrous deoxy alpha-chains, whereas the AHSP/alpha-Hb complex shows reversible hexacoordination of the alpha-Hb without protein aggregation. Upon pressure release, the relaxation kinetics of the transition from the hexacoordinated to pentacoordinated form of alpha-Hb in the presence of AHSP exhibit a biphasic shape. High pressure did not induce dissociation of alpha-Hb from its chaperone, as evidenced by the ligand binding kinetics that show a unique rate for the AHSP/alpha-Hb complex. For both free alpha-Hb and the AHSP/alpha-Hb complex, the bimolecular rate constant of CO binding (k(CO)(on)) versus pressure exhibits a bell shape, attributed to the transition of the rate-determining step from the chemical barrier to the migration of CO within the protein matrix. These results reveal a plasticity of the alpha-Hb active site in the presence of the chaperone and indicate that the AHSP was still active at 300 MPa. The ferric state of the AHSP/alpha-Hb complex shows hexacoordination even at atmospheric pressures, indicating a His-Fe-His binding scheme as previously observed in neuroglobin and cytoglobin. The reaction with hydrogen peroxide of ferric alpha-Hb within the complex also demonstrates a protection against aggregation.     

Internalization of weight bias: Implications for binge eating and emotional well-being

Objective: This study examined the relationship between internalization of negative weight‐based stereotypes and indices of eating behaviors and emotional well‐being in a sample of overweight and obese women. Research Method and Procedures: The sample was comprised of 1013 women who belonged to a national, non‐profit weight loss organization. Participants completed an on‐line battery of self‐report questionnaires measuring frequency of weight stigmatization and coping responses to deal with bias and symptoms of depression and self‐esteem, attitudes about weight and obesity, and binge eating behaviors. In addition, participants were asked to list the most common weight‐based stereotypes and whether they believed them to be true or false. Results: Participants who believed that weight‐based stereotypes were true reported more frequent binge eating and refusal to diet in response to stigma experiences compared with those who reported stereotypes to be false. The degree to which participants believed stereotypes to be true or false was not related to types or amount of stigma experiences reported, self‐esteem, depression, or attitudes toward obese persons. In addition, engaging in weight loss strategies as a response to bias was not predicted by stereotype beliefs or by actual stigma experiences, regardless of the amount or types of stigma reported. Discussion: These findings suggest that obese individuals who internalize negative weight‐based stereotypes may be particularly vulnerable to the negative impact of stigma on eating behaviors and also challenge the notion that stigma may motivate obese individuals to engage in efforts to lose weight. This study highlights a new area of research that warrants attention to better understand weight stigma and its potential consequences for health.     

Speech and non-speech audio-visual illusions: a developmental study

It is well known that simultaneous presentation of incongruent audio and visual stimuli can lead to illusory percepts. Recent data suggest that distinct processes underlie non-specific intersensory speech as opposed to non-speech perception. However, the development of both speech and non-speech intersensory perception across childhood and adolescence remains poorly defined. Thirty-eight observers aged 5 to 19 were tested on the McGurk effect (an audio-visual illusion involving speech), the Illusory Flash effect and the Fusion effect (two audio-visual illusions not involving speech) to investigate the development of audio-visual interactions and contrast speech vs. non-speech developmental patterns. Whereas the strength of audio-visual speech illusions varied as a direct function of maturational level, performance on non-speech illusory tasks appeared to be homogeneous across all ages. These data support the existence of independent maturational processes underlying speech and non-speech audio-visual illusory effects.