Cells employ allosteric regulation as a fundamental mechanism to control critical processes at the molecular level. Computational approaches based on molecular simulations enable in-depth, comparative analyses of allosteric regulation networks, especially when combined with cryo-EM data and biophysical methods such as single molecule FRET (smFRET). The inward rectifier potassium (Kir) channels control the permeation of K+ ions. They are ubiquitously expressed throughout the human body and are important regulators of electrical signaling in the brain, muscle, pancreas, liver, and cardiovascular system. They are also important regulators of salt balance in the kidney. The gating of Kir2.1 is modulated by modulators such as PIP2, anionic lipids, microRNAs (miR). Mutations on Kir2.1 cause several disorders in humans such as Andersen Syndrom (AS). The main objective of KirFlex is to understand the dynamical rearrangement of Kir channels and the allosteric regulation pathways during gating. We will investigate the functional effect of regulators such as PIP2, anionic lipids and miR1 in interaction with Kir2.1 and the impact of two medically important, potentially allosteric Kir2.1 channel mutants: R312H, a loss of function mutant, and M301K, a gain of function mutant. Despite the considerable advances in the biochemical knowledge of the Kir channels, the detailed mechanism by which Kir channels are regulated remains unclear. How mutants cause gating defects is also not known. This project has interdisciplinary experimental and theoretical innovative approaches by the integration of cutting-edge structural (cryo-EM-SPA) and biophysical techniques (smFRET in order to investigate the dynamics of the channel and Surface Plasmon Resonance -SPR). In order to access the many transition states, (which are difficult to access experimentally) we will use in silico methods, particularly a computational methodologies (MDeNM) method recently developed by partner 2. At the conclusion of this project, the structure at high resolution of the human Kir2.1 WT and mutants in complex with modulators such as PIP2, negatively charged lipids, miR1 will be deciphered. The allosteric regulation pathways involved in the gating of the channel and the understanding of the impact of clinically-relevant disease-causing mutations on the structure, dynamics, and function of Kir channels will be unveiled, laying the foundations for future investigation of rationally designed allosteric modulator treatments targeting Kir channels pand articularly developing an innovative class of RNA medicines to treat muscular, cardiac, and bone formation defects associated with AS and other ion channel disorders. The work package of KirFlex are i)..To determine the high-resolution structures of the human Kir2.1 WT and disease-related mutants in complex with PIP2, negatively charged phospholipids, and a specific miR1. ii) To investigate the function and dynamics of these channels (WT and mutants) using smFRET and SPR. iii) To use computational methods to interpret and integrate the experimental results to decipher the allosteric regulation pathways involved in the channel's gating and understand the molecular mechanism of mutant dysfunction. The transdisciplinary consortium assembles two complementary teams with demonstrated expertise in the structure and function of ion channels (Vénien-Bryan C, IMPMC- Sorbonne Université Paris partner 1), and in the integration of experiments and computational methods to study macromolecular dynamics (Pasi M, ENS Paris-Saclay, partner 2). The project is ambitious but remains realistic, considering the partners' expertise and the preliminary results already at hand.

A challenge in modern biology is to understand how circuits of proteins enable cells to monitor information from their environment and make decisions to mount efficient responses. Small GTPases are among the most prominent actors in cellular pathways, and they are involved in the regulation of virtually all cellular processes. A commonality of all small GTPases is their ability to function as molecular switches by their alternation between an inactive, GDP-bound, and an active, GTP-bound form. This simple mechanism is in reality exceedingly complex, because multiple signals converge towards each small GTPase through their numerous activators (guanine nucleotide exchange factors or GEFs, which stimulate their loading with GTP), which in turn generate a unique response by selecting a specific downstream effector that binds to the activated GTPase. Conversely, the level of active GTPases is balanced by the inhibiting activities of their GTPase-activating proteins (GAPs), which inactivate them by stimulating hydrolysis of GTP. Importantly, all these mechanisms operate at the periphery of membranes, which are thus additional components in GTPases signaling. By analogy to telecommunication networks, small GTPases systems can thus be likened to multiplexed molecular circuits, in which multiple inputs are combined into a single signal (the small GTPase) before they are broken down again into specific outputs. Currently, the kinetics and specificity determinants of the small GTPases signaling in this multiplexed framework have remained mysterious. Our hypothesis is that the specificity of signaling arises from multiscale contributions: differential affinities (proteins), molecular compositions (complexes) and spatial partitioning on membranes (nanoclusters). This project addresses this issue by quantitative approaches merging in vitro and in cellulo systems, focusing on the activation and signaling kinetics of the small GTPase Rac1. Rac1 is a master regulator of cell motility, endocytosis and cell growth, and is associated to many pathological conditions such as tumorigenesis, nervous system development disorders, cardiac diseases or infections. This small GTPase is regulated by a very large number of GEFs and GAPs and can signal to diverse effectors, and thus can be seen as archetypal of multiplexing. An important step towards understanding the system-level features of Rac1 signaling in cell biology and disease is the reconstitution of its regulation and activity under well-controlled conditions where kinetics parameters of activation (GDP/GTP exchange and GTP hydrolysis) and signaling (effector binding) can be accurately measured. Bringing together the expertise of the Cherfils lab (ENS Paris-Saclay) in the reconstitution of small GTPases in artificial membranes and of the Coppey lab (Institut Curie Paris) in subcellular quantitative optogenetics, our objective is to identify and quantify these parameters by integrating in vitro kinetics assays using purified proteins and controlled artificial membranes (liposome and supported lipid bilayer) with in cellulo biochemical assays in which regulator concentrations are controled by optogenetics. Ultimately, these ground-truth parameters will be integrated into mathematical models of Rac1 circuitry and signaling. Our project should deliver a molecular « instruction manual » of Rac1 on membranes, pave the way for future studies that address the GTPase multiplexing issue and generate conceptual advances that will broaden our understanding of the many facettes of small GTPases. In the longer term, it may inspire system-based molecular strategies in drug discovery for inhibiting GTPase-controled signaling pathways in diseases.

Streptococcus pneumoniae(PN) is a human nasopharyngeal commensal and major aetiological agent of bacterial meningitis. Our preliminary results indicate that cytotoxicity linked to PN and its major virulence factor pneumolysin (Ply) implicates calcium (Ca2+) signalling mediated by connexins (Cxs), the components of intercellular Gap junctions. Mice infection studies indicate that expression of astroglial Cx43 favoured PN translocation in the brain and meningitis. We also found that Ply-independent secreted factors (PSFs) associated with PN strains causing meningitis trigger Ca2+ signalling and endothelial cell cytotoxicity. The goal of this proposal is to identify and characterize these PSFs, as well as their role and that of Ply in Cxs- and Ca2+-mediated signalling during bacterial crossing of the Blood Brain Barrier (BBB) using a unique reconstituted in vitro model. The role of PSFs in the crossing of the BBB will be characterized in a mice model of PN meningitis.

To invade their host and avoid from being destroyed, intracellular bacterial pathogens inject numerous proteins (collectively called effectors) which exert biochemical functions to take command of host cell pathways. Membrane traffic is among the primary pathways manipulated by these effectors, allowing pathogens to escape from the phago-lysosomal pathway or to convert phagosomes into specialized compartments where they hide and replicate. Understanding the molecular tactics that pathogens use to subvert trafficking machineries is an important issue to elucidating how they survive in the infected cell, which can inspire novel therapeutic strategies to combat infections. The mechanisms of effectors from Legionella pneumophila (Lp) and from the phylogenetically related pathogens Coxiella burnetti (Cb) and Rickettsia prowazekii (Rp), which manipulate membrane traffic are being investigated in this project, using an interdisciplinary approach that combines reconstitution of effectors and their cellular targets on artificial membranes, structural biology by classical and in meso crystallization and cellular microbiology. L. pneumophila is responsible for the Legionnaire’s disease, an acute pneumonia transmitted via ill-maintained water systems. It infects lung macrophages, where it evades destruction by camouflaging in a specialized vacuole that originates from the phagosome and rapidly diverts from the degradative lysosomal pathway by incorporating membranes and proteins from the endoplasmic reticulum. To create this vacuole and persist in it, Lp uses a type IV secretion system to deliver over 280 effectors, a number of which target host cell traffic. A hallmark of Lp is the subversion of Arf and Rab small GTPases (which are chief organizers of membrane traffic) by effectors that mimic host regulators or carry out reversible post-translational modifications. The mechanisms of Lp effectors that manipulate the localization and activity of trafficking GTPases by post-translational modifications is being investigated in this project. C. burnetti is an extremely resistant organism which causes Q-fever, a worldwide disease with acute and chronic stages that is transmitted to humans via aerosols from contaminated soils or livestock. Cb also hijacks trafficking pathways of the cell to establish a specialized membrane-bound organelle, but uses a very different strategy. The Cb-containing vacuole (CCV) derives from fusion of the phagosome with lysosomal vesicles, thus providing the pathogen with an acidic PH environment that enables its intracellular replication. One of the most striking features of the mature CCV is its ability to fuse promiscuously with other lysosome-derived vacuoles in the host cell, which creates a spacious vacuole that contains all of the intracellular bacteria. The structure and function of a novel Cb effector that manipulates autophagy processes to promote the fusogenic properties of the CCV are being investigated in this project. An important function of bacterial effectors that manipulate trafficking pathways is to relocate host GTPases to illegitimate membranes and/or to exclude these GTPases from cellular membranes. The underlying mechanisms rely on their recognition of specific membranes, where they acquire their active structure. Visualizing these interactions and conformational changes at high resolution in the crystal is extremely difficult. The use of in meso phase crystallization to mimic the membrane interface in the crystal is investigated in this project, focusing as a model system on a membrane-regulated effector that activates host GTPases in Legionella and in Rickettsia, the causative agent of epidemic typhus. This ensemble of studies should deliver a high resolution, integrated understanding of the molecular mechanism of effectors that intracellular pathogens use to manipulate membrane traffic and autophagy, as well as new structural methods to study their interactions with membranes.

COupling Ultraquickly and Ultrastrongly Plasmonic and Photonic modes for Largely Efficient Sensing (COUUPPLES). Optical sensors allow contactless interrogation and rely on the availability of numerous sources and detectors. Metal nanoparticles (NPs) are largely used as their localized surface plasmon resonance is altered by small perturbations of their environment, enabling high detection sensitivity. We already demonstrated a spectacular enhancement of the ultrafast optical response of gold NPs once coupled with a resonant photonic mode of a 1D microcavity in the weak-coupling regime, together with a reduction of the resonance linewidth. It is even possible to reach the ultrastrong coupling regime. A laser pulse can induce switching from the strong to the weak coupling in less than a picosecond. Our project will first demonstrate this experimentally by inserting an array of aligned gold nanorods at the photonic antinode of a multi-layered cavity. The anticrossing behaviour of the polariton mode dispersion curve will be shown and the proof for ultrastrong coupling regime will be established. The high susceptibility to the NP environment of the polariton modes and their ultrafast dynamics will then be exploited to realize new plasmon-based sensors with high sensitivity and large effective volume. Hybrid cavities will be elaborated by mixed nanofabrication techniques and their optical response assessed and modelled. The near-field dynamics will be determined by via an original pump-probe fluorescence investigation. The cavities will then be integrated in a microfluidic environment and their potential for sensing will be tested through six different configurations with growing complexity, from the simple continuous monochromatic light interrogation to the exploitation of the spectral and temporal signatures of the device’s ultrafast transient optical response. The sensitivity of these sensing configurations to changes in the refractive index of the gold nanorod environment will be first determined. Then, a DNA aptamer will be grafted on the nanorod surface, able to bind with both large and small biomolecules. In order to establish the proof of concept of our new localized plasmon-based sensing pathways, we will chose as the analyte thrombin, a protein involved in several cardiovascular diseases, as well as small drug molecules possessing known anticancer and anti-viral activities. The project will be carried out through an interdisciplinary approach gathering three academic laboratories: LuMIn for the theoretical and experimental ultrafast optical response assessment, L2n for nanofabrication and optical characterization, and LBPA for biofunctionalization.