Model for the architecture of caveolae based on a flexible, net-like assembly of Cavin1 and Caveolin discs.

Caveolae are invaginated plasma membrane domains involved in mechanosensing, signaling, endocytosis, and membrane homeostasis. Oligomers of membrane-embedded caveolins and peripherally attached cavins form the caveolar coat whose structure has remained elusive. Here, purified Cavin1 60S complexes were analyzed structurally in solution and after liposome reconstitution by electron cryotomography. Cavin1 adopted a flexible, net-like protein mesh able to form polyhedral lattices on phosphatidylserine-containing vesicles. Mutating the two coiled-coil domains in Cavin1 revealed that they mediate distinct assembly steps during 60S complex formation. The organization of the cavin coat corresponded to a polyhedral nano-net held together by coiled-coil segments. Positive residues around the C-terminal coiled-coil domain were required for membrane binding. Purified caveolin 8S oligomers assumed disc-shaped arrangements of sizes that are consistent with the discs occupying the faces in the caveolar polyhedra. Polygonal caveolar membrane profiles were revealed in tomograms of native caveolae inside cells. We propose a model with a regular dodecahedron as structural basis for the caveolae architecture

HSP90-CDC37-PP5 forms a structural platform for kinase dephosphorylation

Activation of client protein kinases by the HSP90 molecular chaperone system is affected by phosphorylation at multiple sites on HSP90, the kinase-specific co-chaperone CDC37, and the kinase client itself. Removal of regulatory phosphorylation from client kinases and their release from the HSP90-CDC37 system depends on the Ser/Thr phosphatase PP5, which associates with HSP90 via its N-terminal TPR domain. Here, we present the cryoEM structure of the oncogenic protein kinase client BRAFV600E bound to HSP90-CDC37, showing how the V600E mutation favours BRAF association with HSP90-CDC37. Structures of HSP90-CDC37-BRAFV600E complexes with PP5 in autoinhibited and activated conformations, together with proteomic analysis of its phosphatase activity on BRAFV600E and CRAF, reveal how PP5 is activated by recruitment to HSP90 complexes. PP5 comprehensively dephosphorylates client proteins, removing interaction sites for regulatory partners such as 14-3-3 proteins and thus performing a ‘factory reset’ of the kinase prior to release

Development of a high-throughput ex-vivo burn wound model using porcine skin, and its application to evaluate new approaches to control wound infection

Biofilm formation in wounds is considered a major barrier to successful treatment, and has been associated with the transition of wounds to a chronic non-healing state. Here, we present a novel laboratory model of wound biofilm formation using ex-vivo porcine skin and a custom burn wound array device. The model supports high-throughput studies of biofilm formation and is compatible with a range of established methods for monitoring bacterial growth, biofilm formation, and gene expression. We demonstrate the use of this model by evaluating the potential for bacteriophage to control biofilm formation by Staphylococcus aureus, and for population density dependant expression of S. aureus virulence factors (regulated by the Accessory Gene Regulator, agr) to signal clinically relevant wound infection. Enumeration of colony forming units and metabolic activity using the XTT assay, confirmed growth of bacteria in wounds and showed a significant reduction in viable cells after phage treatment. Confocal laser scanning microscopy confirmed the growth of biofilms in wounds, and showed phage treatment could significantly reduce the formation of these communities. Evaluation of agr activity by qRT-PCR showed an increase in activity during growth in wound models for most strains. Activation of a prototype infection-responsive dressing designed to provide a visual signal of wound infection, was related to increased agr activity. In all assays, excellent reproducibility was observed between replicates using this mode

Genomic and ecogenomic characterisation of Proteus mirabilis bacteriophage

Proteus mirabilis often complicates the care of catheterized patients through the formation of crystalline biofilms which block urine flow. Bacteriophage therapy has been highlighted as a promising approach to control this problem, but relatively few phages infecting P. mirabilis have been characterized. Here we characterize five phages capable of infecting P. mirabilis, including those shown to reduce biofilm formation, and provide insights regarding the wider ecological and evolutionary relationships of these phages. Transmission electron microscopy (TEM) imaging of phages vB_PmiP_RS1pmA, vB_PmiP_RS1pmB, vB_PmiP_RS3pmA, and vB_PmiP_RS8pmA showed that all share morphologies characteristic of the Podoviridae family. The genome sequences of vB_PmiP_RS1pmA, vB_PmiP_RS1pmB, and vB_PmiP_RS3pmA showed these are species of the same phage differing only by point mutations, and are closely related to vB_PmiP_RS8pmA. Podophages characterized in this study were also found to share similarity in genome architecture and composition to other previously described P. mirabilis podophages (PM16 and PM75). In contrast, vB_PimP_RS51pmB showed morphology characteristic of the Myoviridae family, with no notable similarity to other phage genomes examined. Ecogenomic profiling of all phages revealed no association with human urinary tract viromes, but sequences similar to vB_PimP_RS51pmB were found within human gut, and human oral microbiomes. Investigation of wider host-phage evolutionary relationships through tetranucleotide profiling of phage genomes and bacterial chromosomes, indicated vB_PimP_RS51pmB has a relatively recent association with Morganella morganii and other non-Proteus members of the Morganellaceae family. Subsequent host range assays confirmed vB_PimP_RS51pmB can infect M. morganii

Combined 1H-Detected solid-state NMR spectroscopy and electron cryotomography to study membrane proteins across resolutions in native environments

Membrane proteins remain challenging targets for structural biology, despite much effort, as their native environment is heterogeneous and complex. Most methods rely on detergents to extract membrane proteins from their native environment, but this removal can significantly alter the structure and function of these proteins. Here, we overcome these challenges with a hybrid method to study membrane proteins in their native membranes, combining high-resolution solid-state nuclear magnetic resonance spectroscopy and electron cryotomography using the same sample. Our method allows the structure and function of membrane proteins to be studied in their native environments, across different spatial and temporal resolutions, and the combination is more powerful than each technique individually. We use the method to demonstrate that the bacterial membrane protein YidC adopts a different conformation in native membranes and that substrate binding to YidC in these native membranes differs from purified and reconstituted system

Structural Insights into Viral Determinants of Nematode Mediated Grapevine fanleaf virus Transmission

Many animal and plant viruses rely on vectors for their transmission from host to host. Grapevine fanleaf virus (GFLV), a picorna-like virus from plants, is transmitted specifically by the ectoparasitic nematode Xiphinema index. The icosahedral capsid of GFLV, which consists of 60 identical coat protein subunits (CP), carries the determinants of this specificity. Here, we provide novel insight into GFLV transmission by nematodes through a comparative structural and functional analysis of two GFLV variants. We isolated a mutant GFLV strain (GFLV-TD) poorly transmissible by nematodes, and showed that the transmission defect is due to a glycine to aspartate mutation at position 297 (Gly297Asp) in the CP. We next determined the crystal structures of the wild-type GFLV strain F13 at 3.0 Å and of GFLV-TD at 2.7 Å resolution. The Gly297Asp mutation mapped to an exposed loop at the outer surface of the capsid and did not affect the conformation of the assembled capsid, nor of individual CP molecules. The loop is part of a positively charged pocket that includes a previously identified determinant of transmission. We propose that this pocket is a ligand-binding site with essential function in GFLV transmission by X. index. Our data suggest that perturbation of the electrostatic landscape of this pocket affects the interaction of the virion with specific receptors of the nematode's feeding apparatus, and thereby severely diminishes its transmission efficiency. These data provide a first structural insight into the interactions between a plant virus and a nematode vector

Percolating metallic structures templated on laser-deposited carbon nanofoams derived from graphene oxide: applications in humidity sensing

Carbon nanofoam (CNF) is a low-density, high-surface-area material formed by aggregation of amorphous carbon nanoparticles into porous nanostructures. We report the use of a pulsed infrared laser to prepare CNF from a graphene oxide (GO) target material. Electron microscopy shows that the films consist of dendritic strings that form web-like three-dimensional structures. The conductivity of these structures can be modified by using the CNF as a nanostructured scaffold for gold nanoparticles deposited by sputter coating, controllably increasing the conductivity by up to 4 orders of magnitude. The ability to measure the conductivity of the porous structures allows electrochemical measurements in the environment. Upon decreasing humidity, the pristine CNF exhibits an increase in resistance with a quick response and recovery time. By contrast, the gold-sputtered CNF showed a decrease in resistance, indicating modification of the doping mechanism due to water adsorption. The sensitivity to humidity is eliminated at the percolation threshold of the metal on the CNF

Structural and Functional characterization of the Grapevine fanleaf virus capsid residues required for the transmission by Xiphinema index

La transmission des virus est une étape cruciale du cycle viral. Le Grapevine fanleaf virus (GFLV) et l'Arabis mosaic virus (ArMV), les principaux agents de la maladie du court-noué de la vigne, sont transmis spécifiquement par deux espèces de nématodes, respectivement Xiphinema index et X. diversicaudatum. Des résultats antérieurs ont montré que la protéine de capside (CP) du GFLV détermine sa transmission. Pour caractériser les régions de la CP impliquées dans la vection du GFLV, nous avons combiné génétique inverse, modélisation 3D par homologie, cristallographie aux rayons X et cryomicroscopie électronique (cryo-ME). Plusieurs régions de la CP ont été échangées entre GFLV et ArMV. L'évaluation de la transmission des chimères a montré qu une région de 11 résidus de la CP du GFLV est requise pour sa vection. Par ailleurs, la caractérisation d'un variant spontané du GFLV (TD) faiblement transmis, a révélé l'importance de la mutation Gly297Asp dans la transmission. La structure cristallographique du GFLV (3 °A) et du GFLV-TD (2,7 °A), montre que la perte de transmission du variant a pour origine la présence d'une chaîne latérale chargée négativement exposée à la surface du virus. Ces données suggèrent qu une cavité de surface formée par le domaine B de la CP, serait le site de reconnaissance virus-vecteur. Enfin, la structure de l'ArMV a été obtenue par reconstruction 3D à partir d'images de cryo-ME. La comparaison du GFLV à l'ArMV suggère l'existence de différences structurales au niveau de la cavité. L'ensemble de ce travail ouvre de nouvelles perspectives de recherche visant à comprendre les bases moléculaires de la transmission des Nepovirus par nématodes.Transmission is a key step in the virus life cycle. Grapevine fanleaf virus (GFLV) and Arabis mosaic virus (ArMV), the two major causal agents of grapevine fanleaf disease, are specifically transmitted by Xiphinema index and X. diversicaudatum, respectively. Previous experiments demonstrated the specificity of transmission to be solely determined by the capsid protein (CP). In order to characterize residues within the CP that confer GFLV transmission, we performed a multidisciplinary approach combining reverse genetic, 3D homology modeling, X-ray crystallography and cryoelectron microscopy (cryo-EM). Several GFLV-ArMV chimeric CP were generated and tested for nematodes transmission. This allowed the identification of one region consisting of 11 residues in GFLV transmission. In addition, the characterization of a spontaneous GFLV variant (GFLV-TD) poorly transmitted by X. index, revealed the importance of Gly297Asp mutation in transmission. GFLV and GFLV-TD crystal structures were solved at 3 and 2.7 °A, respectively. Structural comparisons revealed that the near complete loss of GFLV-TD transmission resulted from the single occurrence of a negatively charged side chain highly exposed at the capsid outer surface. These results suggest the virus-vector binding site to consist of a pocket within the CP B domain. Finally, the ArMV structure was determined using cryo-EM and image reconstruction. The comparison with GFLV suggests structural differences on the pocket protrusions. This work opens new research prospects aiming at better understanding the molecular mechanism governing Nepovirus transmission by nematodes