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The original SARS-CoV-2 vaccination regimen (2 doses) induces insufficient short-term response in kidney transplant (KT) recipients. This study assessed the response to a third dose and the long-term immunogenicity after 2 doses in KT.We analyzed the dynamics of the humoral and cellular response by monitoring SARS-CoV-2 IgG antibodies against the Spike-protein (IgG-Spike) and QuantiFERON SARS-CoV-2 IFN-γ release assay 6 mo after the second dose (T2) and 28 d after the third dose of mRNA vaccines (T3) to KT and controls (dialysis patients and healthy individuals).At T2, the percentage of IgG-Spike+ KT and dialysis patients decreased (KT 65.8%-52.6%, hemodialysis 92.6-81.5%, and peritoneal dialysis 100%-90%), whereas 100% of healthy controls remained positive. About the cellular response, the percentage of responders decreased in all groups, especially in KT (22.4%-9.2%, P = 0.081). At T3, 92% of KT, 94%-98% of dialysis patients, and 100% of healthy controls were IgG-Spike+. In terms of antibody titers, patients and controls showed a reduction between T2 and T3 and about 80% of dialysis patients and 100% of controls achieved high titers after the third dose (1479.5 Binding Antibody Units/mL), whereas this percentage was only 50% in KT. With respect to the cellular response, only KT displayed a significant rise after the third dose.The third dose of mRNA vaccine improves both humoral and cellular responses, but less effectively in KT compared with dialysis patients and healthy controls.

G-protein-coupled receptors (GPCRs) are a super-family of membrane proteins and major targets for drug development. However, many GPCRs drug candidates suffer from a lack of selectivity. Photopharmacology gives the possibility of modulating GPCR activity with an unprecedented local and temporal precision with the use of light. In this review, we compile and classify the different strategies in photopharmacology for GPCRs, we revise the different methods of analysis and characterization of light-regulated molecules used for GPCRs, and we give perspective of the impact of photopharmacology in research applications and in the development of new drugs.

Metabotropic glutamate receptors (mGlu) are a family of class C G protein-coupled receptors (GPCRs) with important biological functions and widespread expression. The mechanisms of mGlu activation and the development of allosteric modulators for these dimeric proteins have attracted singular attention including the use of light regulated ligands. Photopharmacology involves the integration of a photoactive moiety into the ligand structure that following specific illumination undergoes a structural rearrangement and changes its biological activity. The use of light-regulated allosteric ligands offers the opportunity to manipulate mGlu signalling with spatiotemporal precision, unattainable with classical pharmacological approaches. In this review, we will discuss some of the innovations that have been made in the allosteric photopharmacology of mGlu receptors to date. We discuss the prospects of these molecular tools in the control of mGluRs and the new perspectives in understanding mGlu mechanisms, pharmacology and (patho)physiology that can ultimately result in innovative drug discovery concepts., The project on which these results are based has received funding from Ministerio de Ciencia e Innovación, Agencia Estatal de Investigación 10.13039/501100011033 and ERDF A way of making Europe (projects I+D+i CTQ2017-89222-R, PCI2018-093047 and PID2020-120499RB-I00), the Neuron-ERANET program (MAGNOLIA project), by the Catalan government (2017 SGR 1604) to AL and XR, and the European Union's Horizon 2020 research and innovation programme under Marie Skłodowska-Curie grant agreement No. 801342 (Tecniospring INDUSTRY, TECSPR19-1-0062) and the Government of Catalonia's Agency for Business Competitiveness (ACCIÓ) to XG-S.

The insecticide carbaryl is commonly found in indirectly exposed freshwater ecosystems at low concentrations considered safe for fish communities. In this study, we showed that after only 24 h of exposure to environmental concentrations of carbaryl (0.066-660 ng/L), zebrafish larvae exhibit impairments in essential behaviours. Interestingly, the observed behavioural effects induced by carbaryl were acetylcholinesterase-independent. To elucidate the molecular initiating event that resulted in the observed behavioural effects, in silico predictions were followed by in vitro validation. We identified two target proteins that potentially interacted with carbaryl, the α2B adrenoceptor (ADRA2B) and the serotonin 2B receptor (HTR2B). Using a pharmacological approach, we then tested the hypothesis that carbaryl had antagonistic interactions with both receptors. Similar to yohimbine and SB204741, which are prototypic antagonists of ADRA2B and HTR2B, respectively, carbaryl increased the heart rate of zebrafish larvae. When we compared the behavioural effects of a 24-h exposure to these pharmacological antagonists with those of carbaryl, a high degree of similarity was found. These results strongly suggest that antagonism of both ADRA2B and HTR2B is the molecular initiating event that leads to adverse outcomes in zebrafish larvae that have undergone 24 h of exposure to environmentally relevant levels of carbaryl., This work was supported by “Agencia Estatal de Investigación” from the Spanish Ministry of Science and Innovation (project PID2020-113371RB-C21), IDAEA-CSIC, Severo Ochoa Centre of Excellence (CEX2018-000794-S), which financed M.F. with Severo Ochoa funds. Juliette Bedrossiantz was supported by a PhD grant (PRE2018-083513) co-financied by the Spanish Government and the European Social Fund (ESF). This work was supported by Ministerio de Ciencia e Innovación, Agencia Estatal de Investigación and ERDF-FEDER European Fund (projects CTQ2017-89222-R and PID2020-120499RB-I00) and by the Catalan Government (2017 SGR 1604 and 2017-SGR-1807) to AL. XR research was financed by the Spanish Ministry of Economy, Industry and Competitiveness (SAF2015-74132-JIN). We thank Dr. Kees Jalink (The Netherlands Cancer Institute, Amsterdam, the Netherlands) for providing the plasmids encoding for the Epac-SH188 biosensor and Dr. Karen Martinez (University of Copenhagen, Copenhagen, Denmark) for providing the HEK 293 SNAP-β1AR). The findings of this report are not to be construed as an official Department of the Army position unless so designated by other authorized documents.

Catecholamine-triggered β-adrenoceptor (β-AR) signaling is essential for the correct functioning of the heart. Although both β

SummaryCatecholamine-triggered β-adrenoceptor (β-AR) signaling is essential for the correct functioning of the heart. Although both β1- and β2-AR subtypes are expressed in cardiomyocytes, drugs selectively targeting β1-AR have proven this receptor as the main target for the therapeutic effects of beta blockers in heart. Here, we report a new strategy for the spatiotemporal control of β1-AR activation by means of light-regulated drugs with a high level of β1-/β2-AR selectivity. All reported molecules allow for an efficient real time optical control of receptor function in vitro. Moreover, using confocal microscopy we demonstrate that the binding of our best hit, pAzo-2, can be reversibly photocontrolled. Strikingly, pAzo-2 also enables a dynamic cardiac rhythm management on alive zebrafish larvae using light, thus highlighting the therapeutic and research potential of the developed photoswitches. Overall, this work provides the first proof of precise control of the therapeutic target β1-AR in native environments using light.

Selective allosteric modulators bear great potential to fine-tune neurotransmitter-induced brain receptor responses. Promising targets are metabotropic glutamate (mGlu) receptors, which are associated to different brain diseases. These multidomain class C GPCRs experience concerted structural rearrangements and rely on allosteric modulation of agonist action to be fully activated. Here we establish live cell compatible fluorescence labeling of mGlu2 by click chemistry through genetic code expansion. Using lanthanide resonance energy transfer, we establish multiple FRET sensors to monitor ligand effects on conformational changes in mGlu2 extracellular domain and subsequently dissect the underlying conformational states by smFRET. Using three distinct FRET sensors, we demonstrate that mGlu activation relies on a ligand-induced sampling of three conformational states. Orthosteric agonists act by promoting the closure of the mGlu2 ligand binding domains, leading to an equilibrium between an inactive intermediate and the active state. Allosteric modulator further push this equilibrium toward the active state, promoting and stabilizing the relative reorientation of the mGlu protomers. These results underline the complex and dynamic nature of such type of neuroreceptors, pointing out that ligands fine-tune activation by differentially acting on the equilibria between multiple states.

Catecholamine-triggered β-adrenoceptor (β-AR) signaling is essential for the correct functioning of the heart. Although both β1 - and β2 -AR subtypes are expressed in cardiomyocytes, drugs selectively targeting β1 -AR have proven this receptor as the main target for the therapeutic effects of beta blockers in the heart. Here, we report a new strategy for the light-control of β1 -AR activation by means of photoswitchable drugs with a high level of β1 -/β2 -AR selectivity. All reported molecules allow for an efficient real-time optical control of receptor function in vitro. Moreover, using confocal microscopy we demonstrate that the binding of our best hit, pAzo-2, can be reversibly photocontrolled. Strikingly, pAzo-2 also enables a dynamic cardiac rhythm management on living zebrafish larvae using light, thus highlighting the therapeutic and research potential of the developed photoswitches. Overall, this work provides the first proof of precise control of the therapeutic target β1 -AR in native environments using light., We thank Ignacio Pérez (IQAC-CSIC, Barcelona), Yolanda Pérez (IQAC-CSIC, Barcelona), Lourdes Muñoz (SimChem, IQAC-CSIC, Barcelona) and Carme Serra (SimChem, IQAC-CSIC, Barcelona) for technical support. We thank Diana Baxter (Institute for Research in Biomedicine, IRB, Barcelona) for her thorough revision of the language of this manuscript. We thank Dr. Kees Jalink (The Netherlands Cancer Institute, Amsterdam, the Netherlands) for providing the plasmids encoding for the Epac-SH188 biosensor. We thank the University of Vic-Central University of Catalonia (UVic-UCC) and Dr. Marta Otero for the material assignment which helped in some biological assays. We thank Nikos Hatzakis for access to the Olympus IX81 confocal microscope (UCPH, DK). This work was supported by ERDF-FEDER European Fund and Ministerio de Ciencia e Innovación, Agencia Estatal de Investigación (projects CTQ2017-89222-R and PID2020-120499RB-I00) and by the Catalan government (2017 SGR 1604) to A.L. X.R. research was financed by the Spanish Ministry of Economy, Industry and Competitiveness (SAF2015-74132-JIN). D.R. research was supported by “Agencia Estatal de Investigación” from the Spanish Ministry of Science and Innovation (project PID2020-113371RB-C21) and IDAEA-CSIC, Severo Ochoa Centre of Excellence (CEX2018-000794-S), which financed M.F. A.D.C. received the support of a fellowship from “la Caixa” Foundation (ID 100010434) under the fellowship code LCF/BQ/DE18/11670012. K.L.M., A.D. and Y.M. were supported by the Novo Nordisk Foundation (NNF20OC0064565).

Simultaneous visualization of the relationship between multiple biomolecules and their ligands or small molecules at the nanometer scale in cells will enable greater understanding of how biological processes operate. We present here high-definition multiplex ion beam imaging (HD-MIBI), a secondary ion mass spectrometry approach capable of high-parameter imaging in 3D of targeted biological entities and exogenously added structurally-unmodified small molecules. With this technology, the atomic constituents of the biomolecules themselves can be used in our system as the “tag” and we demonstrate measurements down to ~30 nm lateral resolution. We correlated the subcellular localization of the chemotherapy drug cisplatin simultaneously with five subnuclear structures. Cisplatin was preferentially enriched in nuclear speckles and excluded from closed-chromatin regions, indicative of a role for cisplatin in active regions of chromatin. Unexpectedly, cells surviving multi-drug treatment with cisplatin and the BET inhibitor JQ1 demonstrated near total cisplatin exclusion from the nucleus, suggesting that selective subcellular drug relocalization may modulate resistance to this important chemotherapeutic treatment. Multiplexed high-resolution imaging techniques, such as HD-MIBI, will enable studies of biomolecules and drug distributions in biologically relevant subcellular microenvironments by visualizing the processes themselves in concert, rather than inferring mechanism through surrogate analyses., Multiplexed ion beam imaging can provide subcellular localisation information but with limited resolution. Here the authors report an ion beam imaging method with nanoscale resolution which they use to assess the subcellular distribution of cisplatin.

Intratumoral variability is a seminal feature of human tumors contributing to tumor progression and response to treatment. Current technologies are unsuitable to accurately track phenotypes and subclonal evolution within tumors, especially in response to genetic manipulations. Here, we developed epitope combinatorial tags (EpicTags), which we coupled to multiplexed ion beam imaging (EpicMIBI) forin situtracking of barcodes within tissue microenvironments. Using this platform, we dissected the spatial component of cell lineages and phenotypes in a xenograft model of small-cell lung cancer. We observed emergent properties from mixed clones leading to the preferential expansion of subclonal patches for both neuroendocrine and non-neuroendocrine cancer cell states in this model. In tumors harboring a fraction of PTEN-deficient cancer cells, we uncovered a non-autonomous increase of subclonal patch size in PTEN wildtype cancer cells. EpicMIBI can facilitatein situinterrogation of cell-intrinsic and cell-extrinsic processes involved in intratumoral heterogeneity.

Intratumoral heterogeneity is a seminal feature of human tumors contributing to tumor progression and response to treatment. Current technologies are still largely unsuitable to accurately track phenotypes and clonal evolution within tumors, especially in response to genetic manipulations. Here, we developed epitopes for imaging using combinatorial tagging (EpicTags), which we coupled to multiplexed ion beam imaging (EpicMIBI) for in situ tracking of barcodes within tissue microenvironments. Using EpicMIBI, we dissected the spatial component of cell lineages and phenotypes in xenograft models of small cell lung cancer. We observed emergent properties from mixed clones leading to the preferential expansion of clonal patches for both neuroendocrine and non-neuroendocrine cancer cell states in these models. In a tumor model harboring a fraction of PTEN-deficient cancer cells, we observed a non-autonomous increase of clonal patch size in PTEN wild-type cancer cells. EpicMIBI facilitates in situ interrogation of cell-intrinsic and cell-extrinsic processes involved in intratumoral heterogeneity.

A thorough understanding of complex spatial host-disease interactions in situ is necessary in order to develop effective preventative measures and therapeutic strategies. Here, we developed Protein And Nucleic acid IN situ Imaging (PANINI) and coupled it with Multiplexed Ion Beam Imaging (MIBI) to sensitively and simultaneously quantify DNA, RNA, and protein levels within the microenvironments of tissue compartments. The PANINI-MIBI approach was used to measure over 30 parameters simultaneously across large sections of archival lymphoid tissues from non-human primates that were healthy or infected with simian immunodeficiency virus (SIV), a model that accurately recapitulates human immunodeficiency virus infection (HIV). This enabled multiplexed dissection of cellular phenotypes, functional markers, viral DNA integration events, and viral RNA transcripts as resulting from viral infection. The results demonstrated immune coordination from an unexpected upregulation of IL10 in B cells in response to SIV infection that correlated with macrophage M2 polarization, thus conditioning a potential immunosuppressive environment that allows for viral production. This multiplexed imaging strategy also allowed characterization of the coordinated microenvironment around latently or actively infected cells to provide mechanistic insights into the process of viral latency. The spatial multi-modal framework presented here is applicable to deciphering tissue responses in other infectious diseases and tumor biology.

This protocol describes how to section formalin-fixed paraffin-embedded (FFPE) tissue samples and mount the tissue sections on gold-coated glass slides for Multiplexed Ion Beam Imaging (MIBI) analysis. This protocol was used to section metastatic breast cancer core biopsies and resections, primary breast cancer resections and neuroblastoma resections for the Human Tumor Atlas Pilot Project (HTAPP).

G protein-coupled receptors (GPCRs) can integrate extracellular signals via allosteric interactions within dimers and higher-order oligomers. However, the structural bases of these interactions remain unclear. Here, we use the GABAB receptor heterodimer as a model as it forms large complexes in the brain. It is subjected to genetic mutations mainly affecting transmembrane 6 (TM6) and involved in human diseases. By cross-linking, we identify the transmembrane interfaces involved in GABAB1-GABAB2, as well as GABAB1-GABAB1 interactions. Our data are consistent with an oligomer made of a row of GABAB1. We bring evidence that agonist activation induces a concerted rearrangement of the various interfaces. While the GB1-GB2 interface is proposed to involve TM5 in the inactive state, cross-linking of TM6s lead to constitutive activity. These data bring insight for our understanding of the allosteric interaction between GPCRs within oligomers., G protein-coupled receptors (GPCRs), such as GABAB, can integrate extracellular signals via allosteric interactions within dimers and oligomers. Here authors use crosslinking and identify two transmembrane interfaces in GABAB which undergo a concerted rearrangement upon agonist activation.

Understanding the mechanisms of HIV tissue persistence necessitates the ability to visualize tissue microenvironments where infected cells reside; however, technological barriers limit our ability to dissect the cellular components of these HIV reservoirs. Here, we developed protein and nucleic acid in situ imaging (PANINI) to simultaneously quantify DNA, RNA, and protein levels within these tissue compartments. By coupling PANINI with multiplexed ion beam imaging (MIBI), we measured over 30 parameters simultaneously across archival lymphoid tissues from healthy or simian immunodeficiency virus (SIV)-infected nonhuman primates. PANINI enabled the spatial dissection of cellular phenotypes, functional markers, and viral events resulting from infection. SIV infection induced IL-10 expression in lymphoid B cells, which correlated with local macrophage M2 polarization. This highlights a potential viral mechanism for conditioning an immunosuppressive tissue environment for virion production. The spatial multimodal framework here can be extended to decipher tissue responses in other infectious diseases and tumor biology.

This document includes information on the MIBI antibody panel used to profile metastatic breast cancer, primary breast cancer and neuroblastoma samples for the Human Atlas Pilot Project (HTAPP). The goal of this panel is mainly focused on studies into the immune response to diseases, such as cancer, and to be able to both phenotype and assign functional readouts to major immune cell types.

A thorough understanding of complex spatial host-disease interactions in situ is necessary in order to develop effective preventative measures and therapeutic strategies. Here, we developed Protein And Nucleic acid IN situ Imaging (PANINI) and coupled it with Multiplexed Ion Beam Imaging (MIBI) to sensitively and simultaneously quantify DNA, RNA, and protein levels within the microenvironments of tissue compartments. The PANINI-MIBI approach was used to measure over 30 parameters simultaneously across large sections of archival lymphoid tissues from non-human primates that were healthy or infected with simian immunodeficiency virus (SIV), a model that accurately recapitulates human immunodeficiency virus infection (HIV). This enabled multiplexed dissection of cellular phenotypes, functional markers, viral DNA integration events, and viral RNA transcripts as resulting from viral infection. The results demonstrated immune coordination from an unexpected upregulation of IL10 in B cells in response to SIV infection that correlated with macrophage M2 polarization, thus conditioning a potential immunosuppressive environment that allows for viral production. This multiplexed imaging strategy also allowed characterization of the coordinated microenvironment around latently or actively infected cells to provide mechanistic insights into the process of viral latency. The spatial multi-modal framework presented here is applicable to deciphering tissue responses in other infectious diseases and tumor biology.

International audience; Glycine receptors (GlyRs) are indispensable for maintaining excitatory/inhibitory balance in neuronal circuits that control reflexes and rhythmic motor behaviors. Here we have developed Glyght, a GlyR ligand controlled with light. It is selective over other Cys-loop receptors, is active in vivo, and displays an allosteric mechanism of action. The photomanipulation of glycinergic neurotransmission opens new avenues to understanding inhibitory circuits in intact animals and to developing drug-based phototherapies.

β

β2-Adrenoceptors (β2-AR) are prototypical G-protein-coupled receptors and important pharmacological targets with relevant roles in physiological processes and diseases. Herein, we introduce Photoazolol-1–3, a series of photoswitchable azobenzene β2-AR antagonists that can be reversibly controlled with light. These new photochromic ligands are designed following the azologization strategy, with a p-acetamido azobenzene substituting the hydrophobic moiety present in many β2-AR antagonists. Using a fluorescence resonance energy transfer (FRET) biosensor-based assay, a variety of photopharmacological properties are identified. Two of the light-regulated molecules show potent β2-AR antagonism and enable a reversible and dynamic control of cellular receptor activity with light. Their photopharmacological properties are opposite, with Photoazolol-1 being more active in the dark and Photoazolol-2 demonstrating higher antagonism upon illumination. In addition, we provide a molecular rationale for the interaction of the different photoisomers with the receptor. Overall, we present innovative tools and a proof of concept for the precise control of β2-AR by means of light., We thank Montserrat Masoliver (UVic-UCC, Vic, Spain), Joan Bertrán (UVic-UCC, Vic, Spain), Jordi Serra (UVic-UCC, Vic, Spain), Lourdes Muñoz (SiMChem, IQAC-CSIC, Barcelona), Maria José Bleda (IQAC-CSIC, Barcelona), Ignacio Pérez (IQAC-CSIC, Barcelona), Yolanda Pérez (IQAC-CSIC, Barcelona) and Carme Serra (SiMChem, IQAC-CSIC, Barcelona) for technical support. We thank Dr. Kees Jalink (The Netherlands Cancer Institute, Amsterdam, the Netherlands) for providing the plasmids encoding for the Epac-SH188 biosensor. We thank the Cisbio Bioassays for their support and technical discussion, as well as for providing plasmids in the preliminary optimization of the biological assays. The project that gave rise to these results received the support of a fellowship from “la Caixa” Foundation (ID 100010434). The fellowship code is (LCF/BQ/DE18/11670012). This work was supported by FEDER/Ministerio de Ciencia, Innovación y Universidades–Agencia Estatal de Investigación (CTQ2017-89222-R), the Catalan government (2017SGR1604), PO FEDER of Catalonia 2014-2020 (project PECT Osona Transformació Social, Ref. 001-P-000382) and the Spanish Ministry of Economy, Industry and Competitiveness (SAF2015-74132-JIN).

Multiplexed ion beam imaging (MIBI) has been previously used to profile multiple parameters in two dimensions in single cells within tissue slices. Here, a mathematical and technical framework for three-dimensional (3D) subcellular MIBI is presented. Ion-beam tomography (IBT) compiles ion beam images that are acquired iteratively across successive, multiple scans, and later assembled into a 3D format without loss of depth resolution. Algorithmic deconvolution, tailored for ion beams, is then applied to the transformed ion image series, yielding 4-fold enhanced ion beam data cubes. To further generate 3D sub-ion-beam-width precision visuals, isolated ion molecules are localized in the raw ion beam images, creating an approach coined as SILM, secondary ion beam localization microscopy, providing sub-25 nm accuracy in original ion images. Using deep learning, a parameter-free reconstruction method for ion beam tomograms with high accuracy is developed for low-density targets. In cultured cancer cells and tissues, IBT enables accessible visualization of 3D volumetric distributions of genomic regions, RNA transcripts, and protein factors with 5 nm axial resolution using isotope-enrichments and label-free elemental analyses. Multiparameter imaging of subcellular features at near macromolecular resolution is implemented by the IBT tools as a general biocomputation pipeline for imaging mass spectrometry., Secondary ion beam mass spectrometry (SIMS) is a method to obtain a chemical snapshot of biological tissue, but the spatial resolution is low. Here, the authors develop a computational and technology pipeline to localise a chemical signal in SIMS in 3D and sub-25 nm accuracy, called Ion Beam Tomography

G protein-coupled receptors (GPCR), including the metabotrobic glutamate 5 receptor (mGlu5), are important therapeutic targets and the development of allosteric ligands for targeting GPCRs has become a desirable approach toward modulating receptor activity. Traditional pharmacological approaches toward modulating GPCR activity are still limited since precise spatiotemporal control of a ligand is lost as soon as it is administered. Photopharmacology proposes the use of photoswitchable ligands to overcome this limitation, since their activity can be reversibly controlled by light with high precision. As this is still a growing field, our understanding of the molecular mechanisms underlying the light-induced changes of different photoswitchable ligand pharmacology is suboptimal. For this reason, we have studied the mechanisms of action of alloswitch-1 and MCS0331; two freely diffusible, mGlu5 phenylazopyridine photoswitchable negative allosteric modulators. We combined photochemical, cell-based, and in vivo photopharmacological approaches to investigate the effects of trans–cis azobenzene photoisomerization on the functional activity and binding ability of these ligands to the mGlu5 allosteric pocket. From these results, we conclude that photoisomerization can take place inside and outside the ligand binding pocket, and this leads to a reversible loss in affinity, in part, due to changes in dissociation rates from the receptor. Ligand activity for both photoswitchable ligands deviates from high-affinity mGlu5 negative allosteric modulation (in the trans configuration) to reduced affinity for the mGlu5 in their cis configuration. Importantly, this mechanism translates to dynamic and reversible control over pain following local injection and illumination of negative allosteric modulators into a brain region implicated in pain control., We thank Carolina Cera and Teresa Sarrias (SimChem, IQAC-CSIC, Barcelona) and Anna Duran, Roser Borràs and Gloria Somalo (MCS group, IQAC-CSIC, Barcelona) for technical support. We thank Maria José Bleda Hernández (IQAC-CSIC, Barcelona) for statistical support. We thank Jean-Philippe Pin (IGF, Montpellier) for scientific support and contributions to this work. We thank Christine Enjalbal (IBMM, Montpellier), Thierry Durroux (IGF, Montpellier), Jean-Louis Banères (IBMM, Montpellier) and Guillaume Lebon (IGF, Montpellier) for scientific discussion. This research was supported by FEDER/Ministerio de Ciencia, Innovación y Universidades–Agencia Estatal de Investigación (CTQ2017-89222-R and PCI2018-093047), the Catalan government (2017SGR1604), by CSIC (PICS program 08212), by Neuron-ERANET, by grants from the Agence Nationale de la Recherche (ANR-16-CE16-0010 and ANR-17-NEU3-0001 under the frame of Neuron Cofund) and by the Programme International de Collaboration Scientifique of the CNRS (PICS 08212). AEB was supported by the Labex EpiGenMed (program « Investissements d’avenir », ANR-10-LABX-12-01).

International audience; Due to the essential roles of glutamate, detection and response to a large range of extracellular concentrations of this excitatory amino acid are necessary for the fine-tuning of brain functions. Metabotropic glutamate receptors (mGluRs) are implicated in shaping the activity of many synapses in the central nervous system. Among the eight mGluR subtypes, there is increasing interest in studying the mGlu3 receptor which has recently been linked to various diseases, including psychiatric disorders. This receptor displays striking functional properties, with a high and, often, full basal activity, making its study elusive in heterologous systems. Here, we demonstrate that Cl− ions exert strong positive allosteric modulation of glutamate on the mGlu3 receptor. We have also identified the molecular and structural determinants lying behind this allostery: a unique interactive “chloride-lock” network. Indeed, Cl− ions dramatically stabilize the glutamate-induced active state of the extracellular domain of the mGlu3 receptor. Thus, the mGlu3 receptors’ large basal activity does not correspond to a constitutive activity in absence of agonist. Instead, it results mostly from a Cl−mediated amplified response to low ambient glutamate concentrations, such as those measured in cell media. This strong interaction between glutamate and Cl− ions allows the mGlu3 receptor to sense and efficiently react to sub-micromolar concentrations of glutamate, making it the most sensitive member of mGluR family.

Glycine receptors (GlyRs) are indispensable to maintain excitatory/inhibitory balance in neuronal circuits controlling reflex and rhythmic motor behaviors. Here we have developed Glyght, the first GlyR ligand controlled with light. It is selective over other cys-loop receptors, active in vivo, and displays an allosteric mechanism of action. The photomanipulation of glycinergic neurotransmission opens new avenues to understand inhibitory circuits in intact animals, and to develop drug-based phototherapies.

Multiplexed ion beam imaging (MIBI) is a method that uses secondary ion mass spectrometry to image antibodies tagged with isotopically pure elemental metal reporters. It is capable of analyzing up to 50 targets simultaneously over a five-log dynamic range. MIBI was developed by Mike Angelo and Sean Bendall in the laboratory of Garry Nolan at Stanford. The current instrument is commercialized by IonPath Inc. The protocol below describes the conjugation of primary antibodies to isotopically pure lanthanides. This process has been extensively described by Guojun Han, et al. in the manuscript Metal-isotope-tagged monoclonal antibodies for high-dimensional mass cytometry.

Multiplexed ion beam imaging (MIBI) is a method that uses secondary ion mass spectrometry to image antibodies tagged with isotopically pure elemental metal reporters. It is capable of analyzing up to 50 targets simultaneously over a five-log dynamic range. MIBI was developed by Mike Angelo and Sean Bendall in the laboratory of Garry Nolan at Stanford. The current instrument is commercialized by IonPath Inc. The protocol below describes the staining of formalin-fixed, paraffin-embedded tissue sections. It was optimized in the Angelo and Bendall labs at Stanford, and further refined in the Nolan lab to its current form.

Multiplexed ion beam imaging (MIBI) has been previously used to profile multiple parameters in two dimensions in single cells within tissue slices. Here, a mathematical and technical framework for three-dimensional subcellular MIBI is presented. We term the approach ion beam tomography (IBT) wherein ion beam images are acquired iteratively across successive, multiple scans and later compiled into a 3D format. For IBT, cells were imaged at 0.2-4 pA ion current across 1,000 axial scans. Consecutive subsets of ion beam images were binned over 3 to 20 slices (above and below) to create a resolved image, wherein binning was incremented one slice at a time to yield an enhanced multi-depth data without loss of depth resolution. Algorithmic deconvolution, tailored for ion beams, was then applied to the transformed ion image series using a hybrid deblurring algorithm and an ion beam current-dependent point-spread function. Three-dimensional processing was implemented by segmentation, mesh, molecular neighborhoods, and association maps. In cultured cancer cells and tissues, IBT enabled accessible visualization of three-dimensional volumetric distributions of genomic regions, RNA transcripts, and protein factors with 65-nm lateral and 5-nm axial resolution. IBT also enabled label-free elemental mapping of cells, allowing “point of source” cellular component measurements not possible for most optical microscopy targets. Detailed multiparameter imaging of subcellular features at near macromolecular resolution should now be made possible by the IBT tools and reagents provided here to open novel venues for interrogating subcellular biology.

Technologies that visualize multiple biomolecules at the nanometer scale in cells will enable deeper understanding of biological processes that proceed at the molecular scale. Current fluorescence-based methods for microscopy are constrained by a combination of spatial resolution limitations, limited parameters per experiment, and detector systems for the wide variety of biomolecules found in cells. We present here super-resolution ion beam imaging (srIBI), a secondary ion mass spectrometry approach capable of high-parameter imaging in 3D of targeted biological entities and exogenously added small molecules. Uniquely, the atomic constituents of the biomolecules themselves can often be used in our system as the “tag”. We visualized the subcellular localization of the chemotherapy drug cisplatin simultaneously with localization of five other nuclear structures, with further carbon elemental mapping and secondary electron visualization, down to ∼30 nm lateral resolution. Cisplatin was preferentially enriched in nuclear speckles and excluded from closed-chromatin regions, indicative of a role for cisplatin in active regions of chromatin. These data highlight how multiplexed super-resolution techniques, such as srIBI, will enable studies of biomolecule distributions in biologically relevant subcellular microenvironments.One Sentence SummaryThree-dimensional multiplexed mass spectrometry-based imaging revealed the subcellular localization of proteins and small molecules at super-resolution.