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.

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.

Protein-protein interactions during the secretion of neurotransmitters and local hormones or events downstream of receptor binding are difficult to study in a physiological context in situ. Although many of the players and their interactions are known, the dynamics of interactions within a cell physiology context are not because they are difficult to measure. One approach is to apply an inhibitory peptide in a spatially localised and time-resolved experiment, measuring the resulting changes in amplitude and time-course of physiological responses. This proposal takes developments in the photochemistry of one or two-photon uncaging and of peptide chemistry, particularly cell penetrating peptides (CPPs), to develop tools for the time resolved dissection of protein-protein interactions during cellular secretion and synaptic transmission. The approach is interdisciplinary, bringing together expertise in one and two-photon excitation, in development and application of CPPs, and cellullar secretion and synaptic transmission to probe protein-protein interactions during intracellular processes. By ‘caging’ critical residues to block binding, inhibitory peptides will be developed that become active only upon photolysis. Because the processes to be targeted are mainly intracellular, CPPs will permit access to the intracellular compartments; the spatial and time-resolved precision will be provided by one or two-photon photolysis in the experimental microscope. To develop specific probes there are 3 technical aspects that need to be optimised and integrated. First, intracellular access of caged peptides is usually by microinjection or perfusion from patch pipettes. CPPs will be developed to permit access of the inactive caged peptide to many cells within a tissue before release of the active peptide by photolysis localised to the structure of interest. Second, caging groups with enhanced two-photon cross-sections will be developed. Third, the caged CPP-peptide inhibitor conjugates will be tested for reduced binding affinity and biological/pharmacological inertness. Cell penetrating peptides. The proposal will develop efficient intracellular delivery for bioactive cargoes, quantifying uptake and mechanisms with MALDI-TOF mass spectrometry. New approaches are CPPs with basic domains functionalised with fatty acids, entering cells mainly by direct translocation. Rigid cyclic peptides will be developed and structures favoring direct translocation selected. Methods of ligation are also being developed to facilitate synthesis. Identifying structural elements that promote cytosolic translocation in combination with functional assays of CPP-caged inhibitors should lead to the development of new CPPs with improved properties. Photochemistry of one and two-photon cages. Caging groups with high one and two photon cross-sections, high water solubility and suitable for protecting amino N-terminal or lysine groups, sulfhydryls, alcohols or carboxylates are being developed around dimethylamino quinoline photochemistry. Endothelial cell secretion. Vascular endothelial cells regulate hemostasis, inflammation, vessel growth and repair and also provide a good model test system in culture for protein interactions during secretion. The molecules involved have been identified and molecular interventions and optical miroscopy are readily applied. Experiments will target syntaxin 2 related peptides, calmodulin inhibitory peptides and PKC inhibitory peptide. Synaptic transmission. Extracellular caged peptide inhibitors of AMPA subtype of glutamate receptor will be used to distinguish fast single site transmission from slow multi synapse spill-over and parasynaptic transmission at specific synapses in the cerebellar cortex. Intracellularly, caged peptides that interfere with receptor localisation at identifiable synapses after photolysis will be used to probe the time-course of changes in synaptic strength during burst stimulation.

Mitochondria constitute a dynamic network whose morphology is conditioned by an equilibrium between fission and fusion events of their membranes. These processes are essential to shape the ultra-structure of the mitochondrial compartment and are thus also crucial for all mitochondrial functions. Consequently, defects in mitochondrial fusion and fission are associated with numerous pathologies. To modulate their membrane dynamics, mitochondria developed an evolutionary conserved strategy that involves large GTPases of the Dynamin-Related Proteins (DRPs) family. While the mechanism by which DRPs promote membrane fission is well understood, how they can also promote mixing of lipid bilayers remains unclear. MITOFUSION thus aims at dissecting how DRPs promote attachment and fusion of mitochondrial outer membranes. For this purpose, a multidisciplinary combination of approaches allying cell and structural biology, biophysics, biochemistry and bio-informatics methods will be employed.

Scaffold proteins assemble and orchestrate cellular events such as the regulation of integral membrane protein activity, e.g. transporters, receptors, and signal transduction pathways. EBP50 (Ezrin-radixin-moesin (ERM)-Binding Phosphoprotein 50)/NHERF1 (NHE3 exchanger Regulatory Factor 1) is a PDZ-scaffold protein highly expressed in liver. At cellular level, EBP50 is localized in the sub-plasma membranous region of epithelial cells along with the cortical actin cytoskeleton. In cell, EBP50 plays a role in epithelial integrity by regulating apical structure and transporters essential for epithelial secretory functions and, in regulation of cell signaling pathways including EGFR. Biliary function is essential for the intestinal absorption of fat and cholesterol homeostasis. Bile primarily formed by the hepatocyte is modified downstream by absorptive and secretory properties of biliary epithelial cells (BEC) in liver and gallbladder (GB). In physiological conditions, both hepatocytes and BEC are virtually quiescent. However, in case of injury, they proliferate in response to diverse regulatory factors which contribute to liver cell mass restoration. Alterations of the absorptive and secretory functions of the biliary system caused by the dysregulation of apical transporters contribute to cholestatic diseases. Therefore, establishment and maintenance of epithelial integrity is crucial to properly ensure the biliary secretory function, which depends on the anchorage and regulation of transporters at the apical domain of liver epithelial cells. EBP50 is expressed by hepatocytes and by BEC along the biliary tree including in the GB in which EBP50 is expressed at higher levels. In hepatocytes and BEC, EBP50 regulates cell surface expression, stability and functions of transporters essential for bile secretory function comprising MRP2, MDR3 and CFTR. Another potentially important regulator of bile composition and volume through an impact on GB epithelium is the membrane G protein-coupled bile acid (BA) receptor TGR5. Interestingly, like EBP50, TGR5 is highly enriched in the GB epithelium and is reported to control CFTR function and expression. However, the precise mechanisms by which TGR5-mediated BA impacts secretory processes in GB have not been explored yet. In particular, potential interactions between EBP50 and TGR5 may couple BA signaling on the one hand, to channels and transporters function and expression on the other hand. Preliminary results strongly suggest a regulatory role of EBP50 in biliary homoeostasis, and therefore will provide the scientific basis of the current project. Mice with targeted EBP50 deletion are viable, develop normally and do not show any gross nor histological signs of liver disease. However, EBP50-KO mice have a significantly larger GB than their WT littermate controls with decreased bile flow, increased plasma total BA concentration and hydrophobicity and, obvious signs of GB inflammation. In line with epithelial hyperplasia observed on GB tissue sections, BEC proliferation is in addition significantly increased in EBP50-KO mice GB. All together, these preliminary data let us to hypothesize that EBP50 regulates biliary homeostasis by acting on hepatobiliary secretion and/or BEC proliferation. Our objective is to study the mechanisms by which EBP50 operates in this context. In addition, cross-talk between EBP50 and TGFR5 will be analyzed since the two proteins are highly expressed in the GB and are both coupled to CFTR and EGFR regulation. Biliary homeostasis will be studied in EBP50-KO mice in basal conditions and in pathophysiological settings during which a biliary adaptive response to BA overload is shown to occur, along with BEC proliferation (e.g. liver regeneration after partial hepatectomy and bile duct-ligation). In a translational perspective, studies on human tumor and non-tumor cholangiopathies will be performed.