827Spatio-Temporal Quantification of FRET in Living Cells by Fast Time-Domain FLIM: A Comparative Study of Non-Fitting Methods

Förster Resonance Energy Transfer (FRET) measured with Fluorescence Lifetime Imaging Microscopy (FLIM) is a powerful technique to investigate spatio-temporal regulation of protein-protein interactions in living cells. When using standard fitting methods to analyze time domain FLIM, the correct estimation of the FRET parameters requires a high number of photons and therefore long acquisition times which are incompatible with the observation of dynamic protein-protein interactions. Recently, non-fitting strategies have been developed for the analysis of FLIM images: the polar plot or “phasor” and the minimal fraction of interacting donor mf_D. We propose here a novel non-fitting strategy based on the calculation of moments. We then compare the performance of these three methods when shortening the acquisition time: either by reducing the number of counted photons N or the number of temporal channels N_ch, which is particularly adapted for the original fast-FLIM prototype presented in this work that employs the time gated approach. Based on theoretical calculations, Monte Carlo simulations and experimental data, we determine the domain of validity of each method. We thus demonstrate that the polar approach remains accurate for a large range of conditions (low N, N_ch or small fractions of interacting donor f_D). The validity domain of the moments method is more restricted (not applicable when f_D<0.25 or when N_ch = 4) but it is more precise than the polar approach. We also demonstrate that the mf_D is robust in all conditions and it is the most precise strategy; although it does not strictly provide the fraction of interacting donor. We show using the fast-FLIM prototype (with an acquisition rate up to 1 Hz) that these non-fitting strategies are very powerful for on-line analysis on a standard computer and thus for quantifying automatically the spatio-temporal activation of Rac-GTPase in living cells by FRET.     

Plasmonic Enhancement by a Continuous Gold Underlayer: Application to SERS Sensing

Plasmonics - In this paper, we report on an improved enhancement of the surface-enhanced Raman scattering (SERS) effect. Such improvement is obtained by using a continuous gold film (underlayer),...     

How to Build a Standardized Country-Specific Environmental Food Database for Nutritional Epidemiology Studies

There is a lack of standardized country-specific environmental data to combine with nutritional and dietary data for assessing the environmental impact of individual diets in epidemiology surveys, which are consequently reliant on environmental food datasets based on values retrieved from a heterogeneous literature. The aim of this study was to compare and assess the relative strengths and limits of a database of food greenhouse gas emissions (GHGE) values estimated with a hybrid method combining input/output and LCA approaches, with a dataset of GHGE values retrieved from the literature. France is the geographical perimeter considered in this study, but the methodology could be applied to other countries. The GHGE of 402 foodstuffs, representative of French diet, were estimated using the hybrid method. In parallel, the GHGE of individual foods were collected from existing literature. Median per-food-category GHGE values from the hybrid method and the reviewed literature were found to correlate strongly (Spearman correlation was 0.83), showing similar rankings of food categories. Median values were significantly different for only 5 (out of 29) food categories, including the ruminant meats category for which the hybrid method gave lower estimates than those from existing literature. Analysis also revealed that literature values came from heterogeneous studies that were not always sourced and that were conducted under different LCA modeling hypotheses. In contrast, the hybrid method helps build reliably-sourced, representative national standards for product-based datasets. We anticipate this hybrid method to be a starting point for better environmental impact assessments of diets.     

Introduced Drosophila subobscura populations perform better than native populations during an oviposition choice task due to increased fecundity but similar learning ability

The success of invasive species is tightly linked to their fitness in a putatively novel environment. While quantitative components of fitness have been studied extensively in the context of invasive species, fewer studies have looked at qualitative components of fitness, such as behavioral plasticity, and their interaction with quantitative components, despite intuitive benefits over the course of an invasion. In particular, learning is a form of behavioral plasticity that makes it possible to finely tune behavior according to environmental conditions. Learning can be crucial for survival and reproduction of introduced organisms in novel areas, for example, for detecting new predators, or finding mates or oviposition sites. Here we explored how oviposition performance evolved in relation to both fecundity and learning during an invasion, using native and introduced Drosophila subobscura populations performing an ecologically relevant task. Our results indicated that, under comparable conditions, invasive populations performed better during our oviposition task than did native populations. This was because invasive populations had higher fecundity, together with similar cognitive performance when compared to native populations, and that there was no interaction between learning and fecundity. Unexpectedly, our study did not reveal an allocation trade‐off (i.e., a negative relationship) between learning and fecundity. On the contrary, the pattern we observed was more consistent with an acquisition trade‐off, meaning that fecundity could be limited by availability of resources, unlike cognitive ability. This pattern might be the consequence of escaping natural enemies and/or competitors during the introduction. The apparent lack of evolution of learning may indicate that the introduced population did not face novel cognitive challenges in the new environment (i.e., cognitive “pre‐adaptation”). Alternatively, the evolution of learning may have been transient and therefore not detected.     

Evolutionary patterns of volatile terpene emissions across 202 tropical tree species

Plant responses to natural enemies include formation of secondary metabolites acting as direct or indirect defenses. Volatile terpenes represent one of the most diverse groups of secondary metabolites. We aimed to explore evolutionary patterns of volatile terpene emission. We measured the composition of damage‐induced volatile terpenes from 202 Amazonian tree species, spanning the angiosperm phylogeny. Volatile terpenes were extracted with solid‐phase micro extraction and desorbed in a gas chromatography–mass spectrometry for compound identification. The chemical diversity of the terpene blend showed a strong phylogenetic signal as closely related species emitted a similar number of compounds. Closely related species also tended to have compositionally similar blends, although this relationship was weak. Meanwhile, the ability to emit a given compound showed no significant phylogenetic signal for 200 of 286 compounds, indicating a high rate of diversification in terpene synthesis and/or great variability in their expression. Three lineages (Magnoliales, Laurales, and Sapindales) showed exceptionally high rates of terpene diversification. Of the 70 compounds found in >10% of their species, 69 displayed significant correlated evolution with at least one other compound. These results provide insights into the complex evolutionary history of volatile terpenes in angiosperms, while highlighting the need for further research into this important class of compounds.     

Influence of Manatees' Diving on Their Risk of Collision with Watercraft

Watercraft pose a threat to endangered Florida manatees (Trichechus manatus latirostris). Mortality from watercraft collisions has adversely impacted the manatee population’s growth rate, therefore reducing this threat is an important management goal. To assess factors that contribute to the risk of watercraft strikes to manatees, we studied the diving behavior of nine manatees carrying GPS tags and time–depth recorders in Tampa Bay, Florida, during winters 2002–2006. We applied a Bayesian formulation of generalized linear mixed models to depth data to model the probability (P_t) that manatees would be no deeper than 1.25 m from the water’s surface as a function of behavioral and habitat covariates. Manatees above this threshold were considered to be within striking depth of a watercraft. Seventy-eight percent of depth records (individual range 62–86%) were within striking depth (mean = 1.09 m, max = 16.20 m), illustrating how vulnerable manatees are to strikes. In some circumstances manatees made consecutive dives to the bottom while traveling, even in areas >14 m, possibly to conserve energy. This is the first documentation of potential cost-efficient diving behavior in manatees. Manatees were at higher risk of being within striking depth in shallow water (<0.91 m), over seagrass, at night, and while stationary or moving slowly; they were less likely to be within striking depth when ≤50 m from a charted waterway. In shallow water the probability of a manatee being within striking depth was 0.96 (CI = 0.93–0.98) and decreased as water depth increased. The probability was greater over seagrass (P_t = 0.96, CI = 0.93–0.98) than over other substrates (P_t = 0.73, CI = 0.58–0.84). Quantitative approaches to assessing risk can improve the effectiveness of manatee conservation measures by helping identify areas for protection.     

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.