The asymmetric distribution of lipids between the two leaflets of cell membranes is a fundamental feature of eukaryotic cells. For instance, while phosphatidylcholine and sphingomyelin are restricted to the outer leaflet of membranes of the late secretory/endocytic pathways in most cell types, phosphatidylserine (PS), phosphatidylethanolamine, and phosphatidylinositol-4,5-bisphosphate are only found in the cytosolic leaflet. Regulated exposure of PS in the outer leaflet of the plasma membrane is an early signal for clearance of apoptotic cells by macrophages or triggering of the blood coagulation cascade. Inside the cell, PS plays critical roles since the high negative surface charge conferred by PS on the cytosolic leaflet of membranes facilitates the recruitment of polybasic motif-containing proteins such as the small GTPase K-Ras and the membrane fission protein EHD1, providing a link between PS distribution and regulation of cell signalling and vesicular trafficking. For transbilayer lipid asymmetry to be maintained, cells have evolved the so-called lipid flippases, transmembrane proteins from the P4-ATPase family which are responsible for the active transport of lipid species from the exoplasmic to the cytosolic leaflet of membranes, at the expense of ATP. Most P4-ATPases require association with transmembrane proteins from the Cdc50 family for proper localization and lipid transport activity. The yeast lipid flippase complex Drs2-Cdc50 has been shown to specifically transport PS and this transport is crucial for bidirectional vesicle trafficking between the endosomal system and the trans-Golgi network (TGN). Mutations in human P4-ATPases have been linked to severe neurological disorders, reproductive dysfunction as well as metabolic and liver disease, underlining the essential role of transbilayer lipid asymmetry in cell physiology. We previously showed, using a combination of limited proteolysis, genetic truncation, and structural approaches, that the catalytic activity of purified Drs2-Cdc50 complex is autoinhibited by its two unstructured N- and C-terminal extensions and activated by phosphatidylinositol-4-phosphate (PI4P). Yet, the molecular mechanism underlying activation of Drs2-Cdc50-dependent lipid transport activity remains unknown. Recently, the small GTPase Arl1 and the Arf-GEF Gea2, a GDP/GTP exchange factor for Arf, were shown to physically interact with the N- and C-termini of Drs2, respectively, and to be required for Drs2-Cdc50-catalyzed lipid transport in isolated TGN vesicles. Arl1 also binds to Gea2, suggesting an intricate mechanism for the regulation of Drs2-mediated transbilayer lipid transport. Based on previous work and our preliminary results, our working hypothesis is that binding of Arl1 and Gea2 to the N- and C-termini of Drs2 relieves autoinhibition and thus activates lipid transport by Drs2-Cdc50. Hence, combining biochemical, in silico and medium/high-resolution structural approaches, FLIPPER aims to dissect this regulatory mechanism, using in vitro reconstitution of the lipid transport machinery. This will be achieved by combining our expertise in the structural and biochemical analysis of small GTPases and Arf-GEFs (J. Cherfils) with structural mass spectrometry techniques, including hydrogen-deuterium exchange mass spectrometry (C. Bechara), structure determination of the Drs2-Cdc50-Arl1-Gea2 complex by cryo-EM (J. Lyons/P. Nissen) and know-how into the biochemistry and functional investigation of lipid flippases (G. Lenoir). Altogether, our proposal aims to provide a mechanistic basis for Drs2 activation in vivo and reveal new functions for understudied small GTPases and large Arf-GEFs such as Arl1 and Gea2.

G quadruplexes (GQs) are non-canonical nucleic acid structures that consist of at least two stacks of G quartets held together by stacking interactions, Hoogsteen base pairs and cation binding. GQ topologies can be categorized based on the relative direction of their four strands. RNA GQs mainly adopt the parallel topology, but non-parallel topologies have also been described. GQ forming sequences are widely distributed in mRNAs and non-coding RNAs, where they regulate gene expression. In vivo, RNA GQs are thought to be largely unfolded, due to the activity of RNA binding proteins (RBP), such as the human helicase DHX36 and the CCHC-type zinc finger nucleic acid binding protein (CNBP). Therefore, RNA GQs are probably transient structures converted by RBPs between their folded and unfolded states. To explore their structure and dynamics as well as their interaction with proteins, a limited set of techniques has been used. While X-ray crystallography and NMR spectroscopy can provide atomically resolved structures, they need high concentrations and give only limited dynamic information (X-ray) or are limited by the molecule size (NMR). Fluorescence techniques are well complementary, being able to monitor molecular interactions and dynamics on a wide time range and at low concentration, but suffering from the need of introducing external labels. Numerous fluorescent purine surrogates have been developed, but they generally destabilize the GQ structure or are highly quenched. In this context, our objective is to validate and apply tzG, an isomorphic fluorescent analogue of G from the isothiazolo[4,3-d]-pyrimidine family, developed by one partner of the project, as a unique tool to site-specifically characterize the conformations, dynamics and molecular interactions of RNA GQs. To reach this aim, we assembled an international multidisciplinary consortium of experts, all highly recognized in their respective fields. The project will be divided in four work packages (WP) and a fifth organizational one (WP0). The aim of WP1 is to synthesize the tzG-labelled GQ forming sequences needed in this project and characterize their structure(s). The effect of tzG on GQ topology and stability will be determined using CD, thermal denaturation experiments and NMR spectroscopy in both K+ and Na+ salt conditions. For sequences with well-resolved NMR spectra, the 3D structure will be then determined, to obtain fine structural details of the impact of tzG insertion in GQ structures. In WP2, we will perform a thorough steady-state and time-resolved fluorescence characterization of the tzG-labelled RNA GQs in both Na+ and K+ salt conditions. Interpretation of the obtained data using a combined MD/QM approach is expected to provide a full picture of the underlying photophysics of tzG in GQs that will be critical to rationalize its spectroscopic properties in GQs and interpret their changes on interaction with RBPs. In WP3, we will validate tzG as a key tool for studying RNA GQs by applying it to decipher the GQ unfolding/refolding mechanism of DHX36 and CNBP proteins, through a combination of NMR, fluorescence spectroscopy and stopped-flow techniques. Finally, using the comprehensive data set of WP1–3, the aim of WP4 will be to refine the methods and interpretative models adopted in the project and, in particular to develop a excitonic Hamiltonian for the study of photoactivated dynamics in multichromophoric systems, including charge transfer processes. Through this study, we anticipate tzG to become the first validated tool to faithfully monitor any substituted G in RNA GQs, and thus lead to breakthroughs in the understanding of GQ properties and RBP mechanisms of action.

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.

Gene expression not only depends on transcription in the nucleus, but also on post-transcriptional events in the cytoplasm, including mRNA degradation, translation, and storage. These processes are governed by the proteins which are bound to mRNAs and by their organization in complexes. They can be associated with particular localizations in ribonucleoprotein granules discovered recently, such as P-bodies. These granules, which are devoid of membrane, contain thousands of mRNAs associated with protein complexes (mRNPs). Their known components indicate their involvement in mRNA degradation, translational repression and RNAi pathway. Yet, so far, there is no evidence of their requirement for these processes, nor any understanding of how they could impact them. Nevertheless, they are present in all eukaryotes, animals and vegetals, from trypanosoma to mammals. The present project proposes to address the question from the granule point of view, by elucidating P-body composition, architecture and mechanism of assembly. Both P-bodies and their components will be studied, using multiscale approaches, made possible by the participation of four partners with distinct and complementary expertises. The coordinator, Dominique Weil, will study P-bodies in their cellular context, and use biochemical, proteomic and transcriptomic approaches to determine their composition. Eric Deprez, will focus on a protein that is essential for P-body assembly, and study the conformation of RNA-protein complexes made in vitro and in vivo by this protein, using biophotonics. Philippe Andrey, will generate a mathematical model of P-bodies, based on electron microscopy experimental data obtained by Gérard Pierron. One part of the project aims at characterizing the mRNP complexes which are present in P-bodies. On one side, as the DEAD-box protein Rck/p54 is essential for P-body assembly in mammals, its protein partners will be characterized, so as to know the distribution of Rck/p54 among mRNA degradation, translational repression, RNAi complexes, or others. The importance of these partners for P-body assembly will be investigated. On the other side, P-bodies will be purified to identify their protein and RNA components. They could contain all types of Rck/p54 complexes or only some, as well as, potentially, unrelated ones. Importantly, it will also elucidate which RNAs are targeted to these granules. A second part of the project aims at characterizing [Rck/p54 – RNA] complexes both in vitro and in vivo. Rck/p54 is an RNA-binding protein which unfolds RNA in vitro. This relaxed conformation of the RNA could play a central role in P-body architecture. Another feature of Rck/p54 is its propensity to homo-oligomerize, which could also participate to granule assembly. Here, we will determine the oligomeric status of RNA-bound Rck/p54, and we will analyze its RNA unfolding activity, in particular in the presence of its main partners. The last part of the project is to better understand P-body architecture using computational models of P-bodies with unfolded RNA and proteins as elementary bricks, and taking into account the new knowledge obtained on P-body content and mRNP conformation. The in silico model will be fitted using EM images of P-bodies in their cellular context, and spatial statistics tools adapted or created for this purpose. This study should provide new insight into P-body architecture and function, including their RNA content. This is of interest not only for these particular granules, but also for the related mRNP granules which are present in particular conditions (stress granules) or particular cell types (germ cells, neurons).

In living organisms, the ability of cells to move across surfaces (cell motility) is a universal feature of multicellularity. In eukaryots, cell motility is essential a different stages of embryogenesis and to maintain an organism’s general homeostasis, for tissue repair or to protect against pathogenic invasions. In bacteria, cell motility is linked to pathogenesis and biofilm formation, which enhances the resistance of the community to environmental insults. Because of their extraordinary resistance to antibiotics and chemicals in general, biofilms pose problems both for public health and the industry. Given their general biological importance, the mechanisms underlying motility regulation have been studied intensively. Remarkably, it appears that the core regulation mechanisms involving small G-proteins of the Ras superfamily is conserved despite the evolutionary distance that separates eukaryots and prokaryots. In eukaryotic systems, these regulations usually involve an arsenal of small G-proteins and their regulators, making it difficult to elucidate the regulatory mechanism. Remarkably the deltaproteobacterium Myxococcus xanthus, uses a single G-protein, MglA, to regulate its direction of movement in response to environmental signals. In this process, MglA associates with GTP and binds to the leading cell pole where it activates the motility machinery. The spatial regulation of MglA depends on a second protein, MglB, a member of the emerging class of Roadblock/Longins of small G-protein regulators, that localizes at the opposite cell pole. At this pole, MglB acts as an MglA GTPase Activating Protein (GAP) and activates the hydrolysis of the MglA-bound GTP, preventing the accumulation of MglA at that pole. Remarkably, this polarity axis formed by MglA and MglB is invertible such that when the localization of MglA and MglB is switched synchronously to the opposite cell poles, the cells stop and resumes movement in the opposite direction (reversal). Reversals are under genetic control and provoked by the signaling activity of the Frz pathway, a bacterial chemosensory-like system, linking environmental changes to motility regulation. The sophisticated experimental tractability of Myxococcus and the relatively low number of regulators makes it a powerful model system to study the regulation of motility at high resolution. However, there are still fundamental questions that need to be solved before an integrated model may be constructed. Starting from preliminary observations, this consortium of three groups with complementary expertise will address three main questions : (i) How does MglB regulate the GTPase activity of MglA and is the mechanism conserved in other Rb-Lg-G-protein pairs? (ii) How does Frz signaling activate the synchronous spatial switching of MglAB? (iii) How is motility regulation by MglAB integrated at the multicellular scale to culminate into cooperative behaviors? Combining reductionist approaches from the realms of structural biology and single cell studies to global approaches such as modeling and genomics, this project aims to define new research directions to understand how autonomous single cell behaviors can be integrated into the cooperation of thousands of individuals, a general question in developmental and evolution biology.