TMEM45A Is Dispensable for Epidermal Morphogenesis,Keratinization and Barrier Formation

TMEM45A gene encodes an initially uncharacterized predicted transmembrane protein. We previously showed that this gene is highly expressed in keratinocytes where its expression correlates with keratinization, suggesting a role in normal epidermal physiology. To test this hypothesis, we generated TMEM45A knockout mice and found that these mice develop without any evident phenotype. The morphology of the epidermis assessed by histology and by labelling differentiation markers in immunofluorescence was not altered. Toluidine blue permeability assay showed that the epidermal barrier develops normally during embryonic development. We also showed that depletion of TMEM45A in human keratinocytes does not alter their potential to form in vitro 3D-reconstructed epidermis. Indeed, epidermis with normal morphogenesis were generated from TMEM45A-silenced keratinocytes. Their expression of differentiation markers quantified by RT-qPCR and evidenced by immunofluorescence labelling as well as their barrier function estimated by Lucifer yellow permeability were similar to the control epidermis. In summary, TMEM45A gene expression is dispensable for epidermal morphogenesis, keratinization and barrier formation. If this protein plays a role in the epidermis, its experimental depletion can possibly be compensated by other proteins in the two experimental models analyzed in this study.     

Crop plants with improved culture and quality traits for food,feed and other uses

The large French research project GENIUS (2012–2019, https://www6.inra.genius-project_eng/) provides a good showcase of current genome editing techniques applied to crop plants. It addresses a large variety of agricultural species (rice, wheat, maize, tomato, potato, oilseed rape, poplar, apple and rose) together with some models (Arabidopsis, Brachypodium, Physcomitrella). Using targeted mutagenesis as its work horse, the project is limited to proof of concept under confined conditions. It mainly covers traits linked to crop culture, such as disease resistance to viruses and fungi, flowering time, plant architecture, tolerance to salinity and plant reproduction but also addresses traits improving the quality of agricultural products for industrial purposes. Examples include virus resistant tomato, early flowering apple and low-amylose starch potato. The wide range of traits illustrates the potential of genome editing towards a more sustainable agriculture through the reduction of pesticides and to the emergence of innovative bio-economy sectors based on custom tailored quality traits.

     

In vivo bioluminescence imaging of Ca signalling in the brain of Drosophila

Many different cells' signalling pathways are universally regulated by Ca(2+) concentration [Ca(2+)] rises that have highly variable amplitudes and kinetic properties. Optical imaging can provide the means to characterise both the temporal and spatial aspects of Ca(2+) signals involved in neurophysiological functions. New methods for in vivo imaging of Ca(2+) signalling in the brain of Drosophila are required for probing the different dynamic aspects of this system. In studies here, whole brain Ca(2+) imaging was performed on transgenic flies with targeted expression of the bioluminescent Ca(2+) reporter GFP-aequorin (GA) in different neural structures. A photon counting based technique was used to undertake continuous recordings of cytosolic [Ca(2+)] over hours. Time integrals for reconstructing images and analysis of the data were selected offline according to the signal intensity. This approach allowed a unique Ca(2+) response associated with cholinergic transmission to be identified by whole brain imaging of specific neural structures. Notably, [Ca(2+)] transients in the Mushroom Bodies (MBs) following nicotine stimulation were accompanied by a delayed secondary [Ca(2+)] rise (up to 15 min. later) in the MB lobes. The delayed response was sensitive to thapsigargin, suggesting a role for intra-cellular Ca(2+) stores. Moreover, it was reduced in dunce mutant flies, which are impaired in learning and memory. Bioluminescence imaging is therefore useful for studying Ca(2+) signalling pathways and for functional mapping of neurophysiological processes in the fly brain.     

Purification and identification of G protein-coupled receptor protein complexes under native conditions

G protein-coupled receptors (GPCRs) constitute the largest family of membrane receptors and are of major therapeutic importance. The identification of GPCR-associated proteins is an important step toward a better understanding of these receptors. However, current methods are not satisfying as only isolated receptor domains (intracellular loops or carboxyl-terminal tails) can be used as "bait." We report here a method based on tandem affinity purification coupled to mass spectrometry that overcomes these limitations as the entire receptor is used to identify protein complexes formed in living mammalian cells. The human MT(1) and MT(2) melatonin receptors were chosen as model GPCRs. Both receptors were tagged with the tandem affinity purification tag at their carboxyl-terminal tails and expressed in human embryonic kidney 293 cells. Receptor solubilization and purification conditions were optimized. The method was validated by the co-purification of G(i) proteins, which are well known GPCR interaction partners but which are difficult to identify with current protein-protein interaction assays. Several new and functionally relevant MT(1)- and MT(2)-associated proteins were identified; some of them were common to both receptors, and others were specific for each subtype. Taken together, our protocol allowed for the first time the purification of GPCR-associated proteins under native conditions in quantities suitable for mass spectrometry analysis.     

Structural Characterization of N-WASP Domain V Using MD Simulations with NMR and SAXS Data

Because of their large conformational heterogeneity, structural characterization of intrinsically disordered proteins (IDPs) is very challenging using classical experimental methods alone. In this study, we use NMR and small-angle x-ray scattering (SAXS) data with multiple molecular dynamics (MD) simulations to describe the conformational ensemble of the fully disordered verprolin homology domain of the neural Aldrich syndrome protein involved in the regulation of actin polymerization. First, we studied several back-calculation software of SAXS scattering intensity and optimized the adjustable parameters to accurately calculate the SAXS intensity from an atomic structure. We also identified the most appropriate force fields for MD simulations of this IDP. Then, we analyzed four conformational ensembles of neural Aldrich syndrome protein verprolin homology domain, two generated with the program flexible-meccano with or without NMR-derived information as input and two others generated by MD simulations with two different force fields. These four conformational ensembles were compared to available NMR and SAXS data for validation. We found that MD simulations with the AMBER-03w force field and the TIP4P/2005s water model are able to correctly describe the conformational ensemble of this 67-residue IDP at both local and global level.     

RAMPs and G protein coupled receptors

RAMPs (receptor activity-modifying proteins) were discovered in 1998 as accessory proteins needed to the functionnal activity of CGRP (calcitonin gene-related peptide) receptors. Three RAMPs generated by three different genes are known in human, rat and mice. The coding sequences of such genes are described, but as yet, regulation sequences are unknown. RAMPs interact with GPCR (G protein-coupled receptors) of class II. In the case of the calcitonin/CGRP peptide family, RAMPs determine the functionnal specificity of the receptor, glycosylate and translocate the receptor to the cell surface. CGRP receptors are observed in presence of the RAMP1/calcitonin receptor-like receptor (CRLR), but the association of RAMP2 or RAMP3 with CRLR generates an adrenomedullin receptor. The calcitonin receptor (CTR) is translocated alone to the cell surface, but interactions of RAMPs with CTR forms amylin receptors. If RAMPs can interact with glucagon, parathyroid hormone and VIP/PACAP (vasoactive intestinal peptide/pituitary adenylate cyclase activating polypeptide (VPACR1)) receptors, the functionnal specificity of these receptors remains unaltered. However, the complex VPACR1/RAMP2 enhances specifically the phosphoinoside signaling pathway.     

Cytological, genetic, and molecular analysis to characterize compatible and incompatible interactions between Medicago truncatula and Colletotrichum trifolii

In this study, a new pathosystem was established using the model plant Medicago truncatula and Colletotrichum trifolii, the causal agent of anthracnose on Medicago sativa. Screening of a few M. truncatula lines identified Jemalong and F83005.5 as resistant and susceptible to Colletotrichum trifolii race 1, respectively. Symptom analysis and cytological studies indicated that resistance of Jemalong was associated with a hypersensitive response of the plant. The two selected lines were crossed, and inoculations with C. trifolii were performed on the resulting F1 and F2 progenies. Examination of the disease phenotypes indicated that resistance was dominant and was probably due to a major resistance gene. Molecular components of the resistance were analyzed through macroarray experiments. Expression profiling of 126 expressed sequence tags corresponding to 92 genes, which were selected for their putative functions in plant defense or signal transduction, were compared in Jemalong and F83005.5 lines. A strong correlation was observed between the number of up-regulated genes and the resistance phenotype. Large differences appeared at 48 h postinoculation; more than 40% of the tested genes were up-regulated in the Jemalong line compared with only 10% in the susceptible line. Interestingly, some nodulin genes were also induced in the resistant line upon inoculation with C. trifolii.