Phagocytosis is a mechanism of internalization and digestion of objects larger than 0.5 microns that relies on receptor triggering leading to actin polymerization and membrane deformation. Partners 1 and 2 have contributed to describe these mechanisms. How multiple receptors simultaneously recognize microbes, pathogens or debris both through direct binding and opsonization, leading to a complex interplay between the signaling pathways and a fine tuning of the fate of the internalised material, is still not well understood. In particular, phagocytosis by C-type lectin receptors that bind carbohydrates residues on the surface of various microorganisms has been overlooked so far. Mannose receptors for instance importantly bind glycoconjugates with terminal mannose, fucose and N-Acetylglucosamine present in bacterial and yeast walls. Partners 2 and 3 developed functionalized lipid droplets coated with tailor-made fluorescent mannolipids to study C type lectin receptors-induced phagocytosis. The complement receptor 3 (CR3) is an integrin that binds microorganisms directly or a complement opsonized-target indirectly. Partner 1 has contributed to dissect signaling associated with CR3. The integrin CR3 was reported to cooperate with Immunoglobulins receptors to ensure efficient phagocytosis, but how different receptors cooperate with each other during signaling, force generation, phagosome formation and maturation, remains to be investigated. This proposal aims at untangling critical steps of phagocytosis taking advantage of a multi-disciplinary approach and unique deformable emulsion droplets coated with fluorescent receptor-targeted ligands as targets for phagocytosis by human macrophages to : - determine by FRET receptors binding and clustering during phagocytosis - monitor directly the forces generated by the phagocyte and identify important regulators of force generation analyse the fate of the internalized material and phagosome maturation upon various receptor engagement, taking advantage of novel functional fluorescent probes. To this end, the complementary expertise of three groups, who have separately made important contributions in their fields and have already collaborated, will be brought together. They will take advantage of a new class of materials, oil-in-water emulsion droplets, developed by Partner 2, which are deformable particles that can be functionalized with biological ligands freely-diffusing at the interface. The interaction and clustering of different receptors in the contact zone between the phagocytic cell and the droplet will be investigated with high spatial resolution using FRET between fluorescent carbohydrate ligands prepared by Partner 3. The deformable droplets are unique tools to directly measure mechanical stresses. The intimate relations between MR and CR3 will be analyzed and the role of potential regulators of the CR3 previously identified by Partner 1 will be tested both on receptor clustering and force generation. To study how receptors influence the fate of the internalized material during phagocytosis, we will combine new probes developed by Partner 3 in various colors allowing multiplexing with the lipid particles. The phagocytosis assays will be performed in primary human macrophages by Partner 1. With this project, we will extend the toolkit to address unsolved questions on phagocytic receptors dynamics in relevant phagocytic cells. We will be able to monitor the underlying mechanobiology of the phagocytosis process, with a novel set of combined expertise and techniques: ligand design, particle formulation, mechanical measurements, time-lapse microscopy and force-dependent integrin partners. Importantly, the receptors of interest play a crucial role in clearance of pathogens as well as neuron pruning, and the phagocytic properties of macrophages can be hijacked in some pathological conditions, which increases the relevance of a better understanding of phagocytosis.

A fundamental understanding of biomolecular events requires knowledge of molecular structure at atomic resolution as well as a detailed description of time-dependent changes in molecular conformation. Indeed, internal motions occurring in biomolecules span a wide range of amplitudes and rates which critically assist them to perform biological functions. Nuclear Magnetic Resonance (NMR) represents a powerful tool to achieve structure/dynamics/function studies of proteins but it suffers from two major drawbacks which are its low sensitivity and the fast nuclear spin relaxation. This strongly limits both the size of the biomolecules that can be studied and the time window for NMR measurements. The CH2PROBE project aims at demonstrating that, as new molecular objects, glycine specifically-labelled proteins will push forward these two limitations. Using innovative NMR experiments, it will be shown that glycine residues offer accurate and unprecedented probes for the study of biological processes. We plan to demonstrate our new developments on the Pin1 protein, a peptidyl-prolyl cis-trans isomerase (PPIase) that plays a major role in various biological processes. Pin1 has already been the subject of intense experimental and computational research over the last two decades and numerous Pin1 ligands have been described. As such, Pin1 will serve as a benchmark for establishing the impact of our NMR developments, in the light of the published literature. Specifically, we will use the glycine residues of Pin1 to enhance the NMR sensitivity even for relatively high molecular weight systems (CH2-TROSY experiments), measure numerous scalar and dipolar couplings (Gly-s3NMR experiments), create long lived nuclear states (LLS) and reveal weakly populated conformations (CEST). These measurements, taken together with MD and DFT calculations, should provide us with a comprehensive view of the protein structure and dynamics. In addition, Pin1 will be studied in complex with ligands that will be designed to offer useful NMR probes and to impact both the protein structure and dynamics. Eventually, the NMR experiments developed in the CH2PROBE project will shed light into subtle conformational features in proteins and will establish the Glycine-NMR approach as a versatile tool for biomolecular studies. These developments will be based on specifically-labelled protein samples, where all the amino acids contain 2H, 12C and 15N nuclei, except for the glycine residues. Since they are responsible for strong dipolar interactions, we will replace all the protons by deuterium in non-Gly residues, whereas 15N spin labels will be kept to allow standard NMR measurements. Such specifically-labelled proteins will be expressed by developing a novel cell-free approach. Once we will demonstrate the efficiency and the selectivity of the protein expression system, it will be possible to specifically incorporate any spin labels at the glycine positions. Indeed, glycine is commercially available with various combinations of NMR active spins, being furthermore the cheapest labelled amino acid. We will take full advantage of this opportunity to optimize the protein samples for each NMR applications. In addition, this new labelling route will allow us to propose innovative D-DNP approaches. For small molecules, signal enhancements by factors up to 50000 have be obtained using D-DNP. We will demonstrate that tremendous sensitivity improvement could be also obtained in proteins, which will pave the way for new applications in biomolecular NMR. The CH2PROBE project will rely on a strong partnership between NMR spectroscopists (Partner 1, UPMC/ENS, Paris), biochemists (Partner 2, IBS, Grenoble), organic chemists (Partner 3, UCP, Cergy-P), and theoretical chemists (Partner 4, ILL, Lyon). Eventually, the developed methods could be applied to any proteins for which structural and dynamics data are lacking or are not accessible using conventional methods.

Bacterial communities colonize and attach to solid surfaces thanks to adhesive molecules exposed on the bacterial outer envelope. While a substantial number of molecular actors involved in bacterial adhesion have been characterized, their dynamics and their coordination on the bacterial envelope remain out of sight because the secretion machineries interfere with the fluorescence of standard probes. Recently, we showed thanks to mechanical assays that adhesive molecules were enriched at the old pole of bacteria. From this polar localization at single cell level, it results that microcolonies composed of rod-shaped bacteria develop into dense aggregates rather than into chains where bacteria would be highly exposed to their environment. This organization at the level of the community has a large impact in terms of biofilm tolerance to antibiotics and causes major health concerns by generating nosocomial diseases. In this project, we propose to use a new generation of fluorescent reporters, in order to measure the spatial dynamics of adhesive proteins exposed on the cell envelope of E. coli. By comparing physical modeling and experiments, we will aim at understanding the microscopic mechanisms that are responsible for adhesion polarity at the single cell level and how antibiotics could perturb this polarity and thus the structure of bacterial communities.

The properties of matter are intimately linked to their nanometric dynamics. Such properties are exploited in many industrial processes (chemical, energy, food, pharmaceutical) and the future successes of these industries depend on their ability to understand, improve and develop new properties. The quest to understand the properties of biological molecules at a fundamental level has been a fascinating field of research for several decades and most questions currently at the forefront of this field are linked to motions around the nanometric scale. Nuclear magnetic resonance (NMR) is a powerful tool to determine motions in complex systems with atomic resolution. Such high resolution is achievable thanks to the use of high magnetic fields. Yet, the determination of site-specific nanosecond motions (particularly slower than 10 ns) by liquid-state NMR is currently almost impossible. This hard limit is due to the necessity to measure relaxation (the rates of return towards equilibrium) at very low magnetic fields, orders of magnitude below the typical fields of high-resolution NMR. In the FASTRELAX-NANODYN project, we propose a radical approach: fast-field-cycling high-resolution relaxometry. We will design and build an innovative instrument as well as develop an experimental and theoretical framework to develop new experiments to probe (sub)nanometric motions at nanosecond timescales. The principle of the method is the following: (1) we obtain high resolution and high sensitivity with a high-field NMR magnet; (2) we use a new generation of sample shuttle to transfer the sample between the high-field center and an second magnet; (3) we rapidly (~1 ms) switch the field of the second magnet to very low magnetic fields (down to ~100 ?T) for relaxation (4) finally, we move the sample back to high fields for detection. The project is built on a small and highly complementary consortium, including the world leader of NMR instrumentation, NMR specialists with diverse expertise, and theoreticians. These methods will be implemented on a series of complex systems from two categories: (1) large protein machine (2) complex fluids relevant for human health, chemical, energy, pharmaceutical and food industries.

Context Striatal cholinergic interneurons (CINs) express vesicular transporters for acetylcholine (VAChT) and glutamate (VGLUT3) and consequently regulate the striatal network with both acetylcholine and glutamate. The synaptic vesicles of CINs have the potential to store and release simultaneously or independently acetylcholine and/or glutamate to regulate striatal activities. Yet, the molecular mechanisms through which striatal cholinergic interneuron releases these two classical neurotransmitters are still largely unknown. In the present proposal, we hypothesize that VAChT and VGLUT3 interact directly or indirectly through partner proteins to regulate acetylcholine/glutamate cotransmission. To address this hypothesis, it is critical to identify the synaptic partners of VAChT and VGLUT3, to determine their localization in synaptic vesicles in relation to VAChT and VGLUT3 and to analyze whether they are involved in acetylcholine and glutamate release. Objectives The central objective of this proposal is to understand the molecular, morphological and functional complexity of acetylcholine/glutamate co-transmission from CINs involving partner proteins of VAChT and VGLUT3. Using a multidisciplinary approach combining computational analyses, super-resolution microscopy, development of new pharmacological tools targeting vesicular glutamate transporters and optogenetics stimulation of CINs coupled to measures of acetylcholine and glutamate release, our ALLEGRO proposal will be organized along three aims: Aim 1. Computational analysis of molecular partners of VAChT and VGLUT3 Using different computational approaches, we will : 1. identify the protein partners of VAChT and VGLUT3 by a cutting-edge large-scale screening of pairs of human protein sequences based on deep learning and by a coevolution analysis addressing the identification of interacting residues between potential partners for VAChT and VGLUT3. 2. reconstruct the protein complexes with recent deep learning approaches for 3D reconstruction. 3. predict the binding sites of the interacting proteins and the modelling through molecular docking of the physical interaction of the VAChT and VGLUT3 domains with identified protein partners. Aim 2. Distribution of the selected partners in VAChT and VGLUT3-expressing synaptic vesicles in CINs and analysis of interactions of partners with VAChT or VGLUT3 Using innovative microscopic approaches, we will : 1. identify which of the partner proteins identified in Aim 1 are present in VAChT and VGLUT3-immunopositive synaptic vesicles by super resolution STED microscopy. 2. measure interactions between partner protein and VAChT or VGLUT3 in synaptic vesicles of cholinergic interneurons by Fluorescence lifetime imaging microscopy combined with Förster resonance energy transfer (FLIM-FRET). Aim 3. Functional role of molecular partners in acetylcholine and glutamate striatal release We will test the causal role of these partners by combining silencing strategies with the monitoring of acetylcholine and glutamate release from striatal cholinergic interneurons. We will : 1. Evaluate the protein partner role on acetylcholine and glutamate uptake on purified synaptic vesicles. 2. Develop glutamate sensors and validate acetylcholine biosensor (m4-based genetically encoded). 3. Characterize acetylcholine and glutamate release from striatal cholinergic interneurons. 4. Determine the effect of silencing of molecular partners on acetylcholine and glutamate release using shRNA. Conclusion This project will thus unravel the molecular actors of neuronal acetylcholine/glutamate cotransmission through the study of acetylcholine/glutamate release from striatal cholinergic interneurons. Beyond its fundamental dimension, the ALLEGRO project should also have profound impact on our understanding of the striatal network whose dysfunction is associated with substance use or eating disorders and possibly accelerates biomarker and therapeutic discovery.