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.

In the course of the ANR-funded ROXANNE (2017-2021) project, we have characterized a novel innate immunity pathway functioning in the control of viral infections in the model organism drosophila. This pathway involves the evolutionarily conserved molecule STING, which activates an NF-kB-dependent transcriptional program that mediates resistance to viral infection. We have recently identified two cGAS-like receptors (cGLRs) involved in the activation of this pathway, through the production of three cyclic dinucleotides (CDNs), 2'3'-cGAMP, 3'2'-cGAMP and 2'3'-c-di-AMP. The goal of this application is to address the new questions raised by this discovery. We will investigate the precise function of the cGLRs in vivo, determining the tissues in which they are active, the cell compartment where they operate and how they integrate their activity with the small interfering (si) RNA pathway, which was until now thought to be the major antiviral mechanism in flies. In particular, we will attempt to identify the ligand activating cGLR2 and the mechanism by which the receptor is activated. In a second aim, we will investigate the function of the three CDNs, addressing their possible role as immunotransmitters in systemic immunity. Finally, we will address the mechanism by which STING activates NF-kB, a question that remains ill characterized in mammals. Overall, this proposal aims at exploiting the resources of the drosophila model to shed light on still poorly understood or emerging aspects of cGAS-STING biology.

Certain plants have the remarkable ability of establishing beneficial root symbioses with soil bacteria to acquire nitrogen, an essential nutrient for their growth. The bacteria provide nitrogen to their host within new specialized root organs, called nodules, formed through integrated multicellular re-differentiation of plant cells. Bacterial entry is a crucial step in this process, and often occurs through the creation of new transcellular apoplastic compartments called infection threads (IT), which guide the symbiotic bacteria towards the developing nodule. These processes must be tightly coordinated, and partner’s exciting new findings suggest that cross-kingdom cell polarisation processes may regulate them. Indeed, through expression and functional studies, they discovered that genes encoding novel DIX-domain plant polarity proteins are linked to nodule formation in various nitrogen-fixing symbioses. DIX-domains are key for plasma membrane association, enabling these proteins to orchestrate PM signal relay via protein-protein interactions. These findings raise the exciting question of whether plants use ancestral DIX-mediated polarization process for nodule ontogeny and bacterial accommodation. Using complementary model legumes (Medicago, Lotus and Mimosa) and non-legume (Parasponia) this project will combine original multi-species functional genomics, phylogenetic foot printing, live microscopy and protein biochemistry strategies to study their roles in the unexplored context of root nodule symbioses. The project should provide innovative results on their ancestral cellular roles, evolutionary recruitment and molecular interactors, and thus shed light on how they organize the infection machinery and nodule formation across different species.