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In the context of global warming, this study aimed to assess the effect of temperature and irradiance on the macroalgal Taonia atomaria holobiont dynamics. We developed an experimental set-up using aquaria supplied by natural seawater with three temperatures combined with three irradiances. The holobiont response was monitored over 14 days using a multi-omics approach coupling algal surface metabolomics and metabarcoding. Both temperature and irradiance appeared to shape the microbiota and the surface metabolome, but with a distinct temporality. Epibacterial community first changed according to temperature, and later in relation to irradiance, while the opposite occurred for the surface metabolome. An increased temperature revealed a decreasing richness of the epiphytic community together with an increase of several bacterial taxa. Irradiance changes appeared to quickly impact surface metabolites production linked with the algal host photosynthesis (e.g. mannitol, fucoxanthin, dimethylsulfoniopropionate), which was hypothesized to explain modifications of the structure of the epiphytic community. Algal host may also directly adapt its surface metabolome to changing temperature with time (e.g. lipids content) and also in response to changing microbiota (e.g. chemical defences). Finally, this study brought new insights highlighting complex direct and indirect responses of seaweeds and their associated microbiota under changing environments., (© 2021 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Background: Although considered as holobionts, macroalgae and their surface microbiota share intimate interactions that are still poorly understood. Little is known on the effect of environmental parameters on the close relationships between the host and its surface-associated microbiota, and even more in a context of coastal pollutions. Therefore, the main objective of this study was to decipher the impact of local environmental parameters, especially trace metal concentrations, on an algal holobiont dynamics using the Phaeophyta Taonia atomaria as a model. Through a multidisciplinary multi-omics approach combining metabarcoding and untargeted LC-MS-based metabolomics, the epibacterial communities and the surface metabolome of T. atomaria were monitored along a spatio-temporal gradient in the bay of Toulon (Northwestern Mediterranean coast) and its surrounding. Indeed, this geographical area displays a well-described trace metal gradient particularly relevant to investigate the effect of such pollutants on marine organisms., Results: Epibacterial communities of T. atomaria exhibited a high specificity whatever the five environmentally contrasted collecting sites investigated on the NW Mediterranean coast. By integrating metabarcoding and metabolomics analyses, the holobiont dynamics varied as a whole. During the occurrence period of T. atomaria, epibacterial densities and α-diversity increased while the relative proportion of core communities decreased. Pioneer bacterial colonizers constituted a large part of the specific and core taxa, and their decrease might be linked to biofilm maturation through time. Then, the temporal increase of the Roseobacter was proposed to result from the higher temperature conditions, but also the increased production of dimethylsulfoniopropionate (DMSP) at the algal surface which could constitute of the source of carbon and sulfur for the catabolism pathways of these taxa. Finally, as a major result of this study, copper concentration constituted a key factor shaping the holobiont system. Thus, the higher expression of carotenoids suggested an oxidative stress which might result from an adaptation of the algal surface metabolome to high copper levels. In turn, this change in the surface metabolome composition could result in the selection of particular epibacterial taxa., Conclusion: We showed that associated epibacterial communities were highly specific to the algal host and that the holobiont dynamics varied as a whole. While temperature increase was confirmed to be one of the main parameters associated to Taonia dynamics, the originality of this study was highlighting copper-stress as a major driver of seaweed-epibacterial interactions. In a context of global change, this study brought new insights on the dynamics of a Mediterranean algal holobiont submitted to heavy anthropic pressures. Video abstract., (© 2021. The Author(s).)

Trace metal (TM) contamination in marine coastal areas is a worldwide threat for aquatic communities. However, little is known about the influence of a multi-chemical contamination on both marine biofilm communities' structure and functioning. To determine how TM contamination potentially impacted microbial biofilms' structure and their functions, polycarbonate (PC) plates were immerged in both surface and bottom of the seawater column, at five sites, along strong TM contamination gradients, in Toulon Bay. The PC plates were incubated during 4 weeks to enable colonization by biofilm-forming microorganisms on artificial surfaces. Biofilms from the PC plates, as well as surrounding seawaters, were collected and analyzed by 16S rRNA amplicon gene sequencing to describe prokaryotic community diversity, structure and functions, and to determine the relationships between bacterioplankton and biofilm communities. Our results showed that prokaryotic biofilm structure was not significantly affected by the measured environmental variables, while the functional profiles of biofilms were significantly impacted by Cu, Mn, Zn, and salinity. Biofilms from the contaminated sites were dominated by tolerant taxa to contaminants and specialized hydrocarbon-degrading microorganisms. Functions related to major xenobiotics biodegradation and metabolism, such as methane metabolism, degradation of aromatic compounds, and benzoate degradation, as well as functions involved in quorum sensing signaling, extracellular polymeric substances (EPS) matrix, and biofilm formation were significantly over-represented in the contaminated site relative to the uncontaminated one. Taken together, our results suggest that biofilms may be able to survive to strong multi-chemical contamination because of the presence of tolerant taxa in biofilms, as well as the functional responses of biofilm communities. Moreover, biofilm communities exhibited significant variations of structure and functional profiles along the seawater column, potentially explained by the contribution of taxa from surrounding sediments. Finally, we found that both structure and functions were significantly distinct between the biofilm and bacterioplankton, highlighting major differences between the both lifestyles, and the divergence of their responses facing to a multi-chemical contamination., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Coclet, Garnier, D’Onofrio, Durrieu, Pasero, Le Poupon, Omanović, Mullot, Misson and Briand.)

The embryonic mouse cortex displays a striking low caudo-medial and high rostro-lateral graded expression of the homeoprotein transcription factor Pax6, which presents both cell autonomous and direct noncell autonomous activities. Through the genetic induction of anti-Pax6 single-chain antibody secretion, we have analyzed Pax6 noncell autonomous activity on the migration of cortical hem- and septum-derived Cajal-Retzius (CR) neurons by live imaging of flat mount developing cerebral cortices. Blocking extracellular Pax6 disrupts tangential CR cell migration patterns by decreasing the distance traveled and changing both directionality and depth at which CR cells migrate. Tracking of single CR cells in mutant cortices revealed that extracellular Pax6 neutralization enhances contact repulsion in medial regions yet reduces it in lateral regions. This study demonstrates that secreted Pax6 controls neuronal migration and distribution and suggests that it acts as a bona fide morphogen at an early stage of cerebral cortex development., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Unraveling the relative importance of both environmental conditions and ecological processes regulating bacterioplankton communities is a central goal in microbial ecology. Marine coastal environments are among the most urbanized areas and as a consequence experience environmental pressures. The highly anthropized Toulon Bay (France) was considered as a model system to investigate shifts in bacterioplankton communities along natural and anthropogenic physicochemical gradients during a 1-month survey. In depth geochemical characterization mainly revealed strong and progressive Cd, Zn, Cu, and Pb contamination gradients between the entrance of the Bay and the north-western anthropized area. On the other hand, low-amplitude natural gradients were observed for other environmental variables. Using 16S rRNA gene sequencing, we observed strong spatial patterns in bacterioplankton taxonomic and predicted function structure along the chemical contamination gradient. Variation partitioning analysis demonstrated that multiple metallic contamination explained the largest part of the spatial biological variations observed, but DOC and salinity were also significant contributors. Network analysis revealed that biotic interactions were far more numerous than direct interactions between microbial groups and environmental variables. This suggests indirect effects of the environment, and especially trace metals, on the community through a few taxonomic groups. These spatial patterns were also partially found for predicted bacterioplankton functions, thus indicating a limited functional redundancy. All these results highlight both potential direct influences of trace metals contamination on coastal bacterioplankton and indirect forcing through biotic interactions and cascading.

Despite clear advances in characterizing marine biofilms, details on their formation and species succession remain scarce particularly during the early stage of development. We investigated the microbial community composition and succession in coastal marine biofilms on plastic. Samples were collected over 75 days of immersion with strengthened samplings during the early stages of biofilm establishment. Biofilm composition was estimated using Illumina Miseq and microbial community interactions were assessed through microbial association network analysis. In silico analyses showed that primers used in most of previous studies considerably underestimated marine biofilm diversity. Unintentionally ignored so far, we showed that Flavobacteriia might be key actors in the functioning of marine biofilms. Gamma-proteobacteria from the genus Oleibacter strongly dominated microbial communities during the first hours of biofilm formation. These pioneer communities were quickly replaced by alpha-proteobacteria and Flavobacteriia. Bacterial communities exhibited fast temporal structure dynamics with taxa displaying rapid increases and declines. A total of 90% of operational taxonomic units (OTUs) were intermittent or ephemeral reinforcing the conclusion that marine biofilms are highly dynamics. With 2/3 of positive significant connections between bacterial OTUs, microbial biofilm communities appear to be more inclined to develop inter-specific cooperation rather than competition and might thus form sets of functional guilds with mutual metabolic exchanges.

In a laboratory study, metal contamination experiments were conducted to investigate the effects of two free copper concentrations (10(-9) and 10(-8) M) on cell growth and on dissolved organic matter exudation by a marine diatom Skeletonema costatum. Throughout incubation, the growth kinetics and exudation of extracellular molecules (i.e. dissolved organic carbon (DOC) and the fluorescent organic matter) were determined. Results revealed an inhibition of S. costatum growth when the free copper level increased (from 10(-9) to 10(-8)). Furthermore, DOC release was more significant in cultures contaminated by 10(-9) M Cu(2+) than in control, suggesting a coping mechanism developed by this species. In this study, samples were daily analysed by 3D-fluorescence and PARAFAC algorithm, in order to compare the fluorescent material produced during growth under different contaminations. PARAFAC treatment revealed two main contributions: one related to the biological activity (C1), the other linked to the marine organic matter (C2). The third component C3 was typically protein-like. This fluorophore was considered as a tryptophan-like fluorophore, whereas the C1 and the C2 components were associated to marine production such as humic matter.

Homeoprotein transcription factors play fundamental roles in development, ranging from embryonic polarity to cell differentiation and migration. Research in recent years has underscored the physiological importance of homeoprotein intercellular transfer in eye field development, axon guidance and retino-tectal patterning, and visual cortex plasticity. Here, we have used the embryonic chick neural tube to investigate a possible role for homeoprotein Pax6 transfer in oligodendrocyte precursor cell (OPC) migration. We report the extracellular expression of Pax6 and the effects of gain and loss of extracellular Pax6 activity on OPCs. Open book cultures with recombinant Pax6 protein or Pax6 blocking antibodies, as well as in ovo gene transfer experiments involving expression of secreted Pax6 protein or secreted Pax6 antibodies, provide converging evidences that OPC migration is promoted by extracellular Pax6. The paracrine effect of Pax6 on OPC migration is thus a new example of direct non-cell autonomous homeoprotein activity.

The crystal structure of the extracellular domain (ECD) of the pentameric ligand-gated ion-channel from Gloeobacter violaceus (GLIC) was solved at neutral pH at 2.3 A resolution in two crystal forms, showing a surprising hexameric quaternary structure with a 6-fold axis replacing the expected 5-fold axis. While each subunit retains the usual beta-sandwich immunoglobulin-like fold, small deviations from the whole GLIC structure indicate zones of differential flexibility. The changes in interface between two adjacent subunits in the pentamer and the hexamer can be described in a downward translation by one inter-strand distance and a global rotation of the second subunit, using the first one for superposition. While global characteristics of the interface, such as the buried accessible surface area, do not change very much, most of the atom-atom interactions are rearranged. It thus appears that the transmembrane domain is necessary for the proper oligomeric assembly of GLIC and that there is an intrinsic plasticity or polymorphism in possible subunit-subunit interfaces at the ECD level, the latter behaving as a monomer in solution. Possible functional implications of these novel structural data are discussed in the context of the allosteric transition of this family of proteins. In addition, we propose a novel way to quantify elastic energy stored in the interface between subunits, which indicates a tenser interface for the open form than for the closed form (rest state). The hexameric or pentameric forms of the ECD have a similar negative curvature in their subunit-subunit interface, while acetylcholine binding proteins have a smaller and positive curvature that increases from the apo to the holo form., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Pentameric ligand-gated ion channels from the Cys-loop family mediate fast chemo-electrical transduction, but the mechanisms of ion permeation and gating of these membrane proteins remain elusive. Here we present the X-ray structure at 2.9 A resolution of the bacterial Gloeobacter violaceus pentameric ligand-gated ion channel homologue (GLIC) at pH 4.6 in an apparently open conformation. This cationic channel is known to be permanently activated by protons. The structure is arranged as a funnel-shaped transmembrane pore widely open on the outer side and lined by hydrophobic residues. On the inner side, a 5 A constriction matches with rings of hydrophilic residues that are likely to contribute to the ionic selectivity. Structural comparison with ELIC, a bacterial homologue from Erwinia chrysanthemi solved in a presumed closed conformation, shows a wider pore where the narrow hydrophobic constriction found in ELIC is removed. Comparative analysis of GLIC and ELIC reveals, in concert, a rotation of each extracellular beta-sandwich domain as a rigid body, interface rearrangements, and a reorganization of the transmembrane domain, involving a tilt of the M2 and M3 alpha-helices away from the pore axis. These data are consistent with a model of pore opening based on both quaternary twist and tertiary deformation.

Food odours are major determinants for food choice; their detection is influenced by nutritional status. Among different metabolic signals, insulin plays a major role in food intake regulation. The aim of the present study was to investigate a potential role of insulin in the olfactory mucosa (OM), using ex vivo tissues and in vitro primary cultures. We first established the expression of insulin receptor (IR) in rat olfactory mucosa. Transcripts of IR-A and IR-B isoforms, as well as IRS-1 and IRS-2, were detected in OM extracts. Using immunocytochemistry, IR protein was located in olfactory receptor neurones, sustentacular and basal cells and in endothelium of the lamina propria vessels. Moreover, the insulin binding capacity of OM was quite high compared to that of olfactory bulb or liver. Besides the main pancreatic insulin source, we demonstrated insulin synthesis at a low level in the OM. Interestingly 48 h of fasting, leading to a decreased plasmatic insulin, increased the number of IR in the OM. Local insulin concentration was also enhanced. These data suggest a control of OM insulin system by nutritional status. Finally, an application of insulin on OM, aiming to mimic postprandial insulin increase, reversibly decreased the amplitude of electro-olfactogramme responses to odorants by approximately 30%. These data provide the first evidence that insulin modulates the most peripheral step of odour detection at the olfactory mucosa level.

Perception of thousands of odors by a few hundreds of olfactory receptors (ORs) results from a combinatorial coding, in which one OR recognizes multiple odorants and an odorant is recognized by a specific group of ORs. Moreover, odorants could act both as agonists or antagonists depending on the OR. This dual agonist-antagonist combinatorial coding is in good agreement with behavioral and psychophysical observations of mixture perception. We previously described the odorant repertoire of a human OR, OR1G1, identifying both agonists and antagonists. In this paper, we performed a 3D-quantitative structure-activity relationship (3D-QSAR) study of these ligands. We obtained a double-alignment model explaining previously reported experimental activities and permitting to predict novel agonists and antagonists for OR1G1. These model predictions were experimentally validated. Thereafter, we evaluated the statistical link between OR1G1 response to odorants, 3D-QSAR categorization of OR1G1 ligands, and their olfactory description. We demonstrated that OR1G1 recognizes a group of odorants that share both 3D structural and perceptual qualities. We hypothesized that OR1G1 contributes to the coding of waxy, fatty, and rose odors in humans.

Ligand-gated ion channels (LGICs) mediate excitatory and inhibitory transmission in the nervous system. Among them, the pentameric or 'Cys-loop' receptors (pLGICs) compose a family that until recently was found in only eukaryotes. Yet a recent genome search identified putative homologues of these proteins in several bacterial species. Here we report the cloning, expression and functional identification of one of these putative homologues from the cyanobacterium Gloeobacter violaceus. It was expressed as a homo-oligomer in HEK 293 cells and Xenopus oocytes, generating a transmembrane cationic channel that is opened by extracellular protons and shows slow kinetics of activation, no desensitization and a single channel conductance of 8 pS. Electron microscopy and cross-linking experiments of the protein fused to the maltose-binding protein and expressed in Escherichia coli are consistent with a homo-pentameric organization. Sequence comparison shows that it possesses a compact structure, with the absence of the amino-terminal helix, the canonical disulphide bridge and the large cytoplasmic domain found in eukaryotic pLGICs. Therefore it embodies a minimal structure required for signal transduction. These data establish the prokaryotic origin of the family. Because Gloeobacter violaceus carries out photosynthesis and proton transport at the cytoplasmic membrane, this new proton-gated ion channel might contribute to adaptation to pH change.

Using fluorescence lifetime microspectroscopy and imaging techniques, we have studied the fluorescence of cyan fluorescent protein (CFP) transiently expressed in HEK-293 cells, in the presence or absence of its fluorescence resonance energy transfer (FRET) partner, yellow fluorescent protein (YFP). When the two proteins are attached through a 27-amino-acid linker, a 33 % average efficiency of intramolecular energy transfer is accurately determined inside the cell. Additionally, we observe a systematic quenching of the CFP fluorescence with increasing levels of protein expression. This quenching cannot be accounted for by formation of the previously described dimer of GFP-related proteins, since its magnitude is unchanged when the fluorescent proteins carry the mutation A206K shown to dissociate this dimer in vitro. Even when the intracellular protein concentration largely exceeds the in vitro dissociation constant of the dimer, self-association remains undetectable, either between free proteins or intramolecularly within the CFP-YFP construct. Instead, the detailed concentration effects are satisfactorily accounted for by a model of intermolecular, concentration-dependent energy transfer, arising from molecular proximity and crowding. In the case of CFP alone, we suggest that self-quenching could result from a pseudo-homo FRET mechanism between different, spectrally shifted emissive forms of the protein. These phenomena require careful consideration in intracellular FRET studies.

The nicotinic acetylcholine receptors (nAChRs) and the 5-HT3 serotonin receptor subtype belong to a superfamily of neurotransmitter-gated ion channels involved in fast synaptic communication throughout the nervous system. Their trafficking to the neuron plasmalemma, as well as their targeting to specific subcellular compartments, is critical for understanding their physiological role. In order to investigate the cellular distribution of these receptors, we tagged the N-termini of alpha3beta4-nAChR subunits and the 5-HT3AR subunit with cyan and yellow fluorescent proteins (CFP, YFP). The fusion subunits were coexpressed in human embryonic kidney (HEK-293) cells, where they assemble into functional receptor channels, as well as in primary cultures of hippocampal neurons. Fluorescence microscopy of living cells revealed that the heteropentameric alpha3CFP-beta4 and YFP-alpha3beta4 receptors are mainly distributed in the endoplasmic reticulum, while the homopentameric YFP-5-HT3A receptor was localized both to the plasma membrane and within intracellular compartments. Moreover, the YFP-5-HT3A receptor was found to be targeted to the micropodia in HEK-293 cells and to the dendritic spines in hippocampal neurons, where it could be accessed by extracellularly applied specific fluorescent probes. The efficient targeting of the YFP-5-HT3A to the cytoplasmic membrane is in line with the large serotonin-elicited currents (nA range) measured by whole-cell voltage-clamp recordings in transfected HEK-293 cells. In contrast, alpha3beta4-nAChRs expressed in the same cells yielded weaker ACh-evoked responses. Taken together, the fluorescent and electrophysiological studies presented here demonstrate the predominant intracellular location of alpha3beta4-nACh receptors and the predominant expression of the 5-HT3AR in dendritic surface loci.