The cell wall is important for bacterial survival and shape, and its biosynthetic mechanism is the target of antibiotics such as beta-lactams and vancomycin. The spread of resistant strains, however, has thwarted the usefulness of these drugs and calls for efforts towards the understanding of processes that could lead to innovative treatments. One of the main pathogens signaled by the WHO as urgently necessitating novel treatment approaches is Pseudomonas aeruginosa, a Gram-negative organism that is the causative agent of hospital- and community-acquired infections, in addition to being a major threat for cystic fibrosis patients. In BAC-ASSEMBLY, we will continue our very successful collaboration in the study of the P. aeruginosa cell wall and will tackle the study of a cell wall-forming complex encompassing cytoplasmic, membrane, and periplasmic proteins. Thanks to an ANR grant (PSEUDO-WALL) that also supported this partnership, Partners 1 and 2 took major steps forward in this field by structurally and functionally characterizing, for the first time, the oligomeric forms of the P. aeruginosa peptidoglycan scaffolding factor, MreC. These totally novel results suggested how such assemblies could participate in turning cell wall biosynthesis on/off, thus playing a key role in its regulation. In BAC-ASSEMBLY, we will go beyond these initial successful results, expanding our effort to characterize a three-compartment, multi-partner cell wall formation complex from P. aeruginosa using X-ray crystallography, electron microscopy, biochemistry, and microbiology techniques. Lastly, we will employ a genome-wide screen in a clinical strain to identify additional partners and regulators of this central P. aeruginosa cell wall formation complex, providing a fresh outlook on the search for new antimicrobials.

The "endosomal sorting complex required for transport" (ESCRT) catalyzes a wide range of physiological and pathological membrane remodeling processes including budding of vesicles and enveloped viruses, cell division and many others involving topologically similar inside-out budding processes. The eukaryotic ESCRT machinery is composed of 5 complexes, ESCRT-0, ESCRT-I, ESCRT-II, ESCRT-III and the ATPase VPS4. ESCRT-III and VPS4 constitute the machinery that executes membrane fission and both are recruited to all ESCRT-catalyzed remodeling processes. Evidence suggests that ESCRT-III members CHMP4, CHMP3 and CHMP2 play an essential role in membrane constriction and fission and could represent with VPS4 a minimal fission machinery. Because ESCRTs operate inside membrane necks, the question how ESCRT filaments organize to achieve membrane constriction and cleavage in conjunction with VPS4 remains a major challenge. The objective of our proposal is to understand the structural basis and the mechanisms of ESCRT-III and VPS4-catalyzed membrane remodeling processes leading to membrane fission. ESCRTs operate in systems such as the cytokinetic midbody that requires constriction from a large diameter (µm) down to complete constriction and membrane fission while vesicle or enveloped virus budding requires membrane neck constriction starting from smaller diameters of about 50 nm. Here we will focus on the reconstitution of constriction and membrane fission from smaller diameters resembling those of vesicles or enveloped viruses. We hypothesize that functional ESCRT-III recruitment/polymerization on membranes requires optimal membrane geometries such as a full catenoid (dumb-bell) or a truncated one, like in a budding tube. The aim of NECK4FISSION is to set up model membranes shapes that recapitulate native membrane neck structures to allow reconstitution of membrane fission with a minimal set of ESCRTs and VPS4. We will develop four different in vitro systems to reconstitute ESCRT-III/VPS4 function. Two are based on membrane tubes preformed around ESCRT-III polymers or pulled from GUVs and two systems are based on the reconstitution of artificial virus-like budding systems employing GUVs and LUVs. We will employ high-resolution imaging techniques and micromanipulation to establish conditions for fission and we will devise state of the art cryo-EM imaging to visualize the effect of ESCRT-IIIs and VPS4 on membrane tubes and on the neck of membrane buds wrapped around virus-like particles inside LUVs and GUVs. These in vitro assays will be complemented by Cryo-TM imaging of HIV-1 budding site employing FIB-SEM and cryo-ET. Finally, all data will be considered to develop a novel membrane fission physical model based on elastic coupling of the ESCRT machinery to the local membrane shape and on membrane viscous stresses created by ATP-dependent remodeling of ESCRT-III, which has not been considered yet. Thus, we will unveil how ESCRT-III and VPS4 contribute to membrane deformation and mechanical stress under different geometrical constraints. Together our data will provide novel important insight into the function, structure and dynamics of ESCRT-III/VPS4-catalyzed membrane fission and on the underlying mechanisms.

Eukaryotic transcription produces large amounts of RNA, with some being processed and exported to the cytoplasm, while others are aberrant and eliminated in the nucleus. The nuclear cap-binding complex (CBC) plays a pivotal role in these processes. During early transcription, RNA-bound CBC forms a complex with the ARS2 protein (CBCA). To influence the fate of transcripts, various proteins, referred to as RNA 'effectors', compete for interactions with CBCA to ultimately direct transcripts towards processing, export, or degradation. Among them are PHAX, notably responsible for the nuclear export of snRNAs, as well as NCBP3 and hnRNPC, involved in mRNA export. However, the molecular mechanisms governing the competition between these effectors, and their organisation within CBC higher order complexes remain unclear. The primary goal of the project is to characterize, structurally and functionally, these essential CBC-mediated RNA sorting mechanisms, focusing on the role of PHAX, NCBP3 and hnRNPC. To achieve this ambitious goal, the project will rely on a collaborative effort of three partners with complementary expertise and promising preliminary results. The proposed work will thus expand our understanding of this fundamental regulatory mechanism in eukaryotic gene expression.

Moving to cryogenic temperature is an important step in the super-resolution fluorescence microscopy field, to overcome artifacts resulting from chemical fixation and to facilitate cryo-correlative studies. Yet, despite significant progress in the last years, cryo-nanoscopy and, in particular, cryo-SMLM, has not provided super-resolved images of sufficiently high quality. A major obstacle to obtaining truly high-resolution cryo-SMLM images is the altered photophysical behavior of fluorophores adapted to SMLM, which are not able to properly photoswitch anymore below the so-called “glass-transition” temperature. This proposal focuses on the optimization of cryo-SMLM using fluorescent proteins (FPs) as markers. Our goal is to significantly improve the quality of achievable FP-based cryo-SMLM images by (i) engineering and better understanding the photophysical properties of various FPs at cryogenic temperature, (ii) modifying a cryo-SMLM microscope to collect better data and (iii) developing the nuclear pore complex (NPC) as a metrology tool to quantitatively evaluate cryo-SMLM performance. In (i), using rsEGFP2 and mEmerald, and thanks to a combination of X-ray crystallography, microspectrophotometry, EPR and single-molecule imaging, we will investigate the nature of the cryo-switched-off states, notably the role of the triplet state and radical states. To boost cryo-switching, we will manipulate these states with UV and IR light. We will also attempt to improve cryo-switching of cyan and red FPs for future multicolor cryo-SMLM. In (ii), we will upgrade our cryo-SMLM microscope for stability, light collection efficiency and flexible illumination with UV and IR light. In (iii) we will use the nuclear pore complex and CRISPR-Cas9 genome editing to assess quantitatively cryo-SMLM performance with our best-performing FPs and under optimized data-collection and data-processing strategies. CryoQN will increase knowledge of FPs beyond state-of-the-art and push cryo-nanoscopy towards quantitative measurements.

In prokaryotes, the frequency of amino acids in proteomes is strongly correlated with the optimal growth temperature (OGT), but the associated substitutional patterns and their dynamics remain poorly understood. The ThermAdapt project addresses these two important issues through an original approach combining genomics, ancestral sequence reconstructions, structural modeling, experimental biology, and persistent homology. The ThermAdapt projects aims at: 1- Tracing back the evolution of OGTs and associated substitutional patterns in different branches of the Tree of Life. The collected data will allow to determine whether substitutional patterns are universal or lineages-specific. 2- Comparing substitutions associated to similar but independant OGT shifts in proteins families, in order to determine whether substitutions occurr at random or specific positions in protein sequences, and thus whether the thermoadaptation process is mainly stochastic or deterministic. 3- Studying the impact of substitutions on the structure and function of proteins by molecular modeling. These predictions will be experimentally tested. To propose new algorithms to model thermoadaptation processes in the primary sequences of proteins as well as in their 3D structures, and in time, using Bayesian and persistent homology approaches. Altogether, the ThermAdapt project will improve our knowledge of mechanisms and patterns of thermoadaptation in prokaryotes.