The membrane-associated form of as1- casein interacts with cholesterol-rich detergent-resistant microdomains

Caseins, the main milk proteins, interact with colloidal calcium phosphate to form the casein micelle. The mesostructure of this supramolecular assembly markedly influences its nutritional and technological functionalities. However, its detailed molecular organization and the cellular mechanisms involved in its biogenesis have been only partially established. There is a growing body of evidence to support the concept that as1-casein takes center stage in casein micelle building and transport in the secretory pathway of mammary epithelial cells. Here we have investigated the membrane-associated form of as1-casein in rat mammary epithelial cells. Using metabolic labelling we show that as1-casein becomes associated with membranes at the level of the endoplasmic reticulum, with no subsequent increase at the level of the Golgi apparatus. From morphological and biochemical data, it appears that caseins are in a tight relationship with membranes throughout the secretory pathway. On the other hand, we have observed that the membrane-associated form of as1-casein co-purified with detergent-resistant membranes. It was poorly solubilised by Tween 20, partially insoluble in Lubrol WX, and substantially insoluble in Triton X-100. Finally, we found that cholesterol depletion results in the release of the membrane-associated form of as1-casein. These experiments reveal that the insolubility of as1-casein reflects its partial association with a cholesterolrich detergent-resistant microdomain. We propose that the membrane-associated form of as1-casein interacts with the lipid microdomain, or lipid raft, that forms within the membranes of the endoplasmic reticulum, for efficient forward transport and sorting in the secretory pathway of mammary epithelial cells

Capsicumicine, a new bioinspired peptide from red peppers prevents staphylococcal biofilm in vitro and in vivo via a matrix anti-assembly mechanism of action

Staphylococci are pathogenic biofilm-forming bacteria and a source of multidrug resistance and/or tolerance causing a broad spectrum of infections. These bacteria are enclosed in a matrix that allows them to colonize medical devices, such as catheters and tissues, and that protects against antibiotics and immune systems. Advances in antibiofilm strategies for targeting this matrix are therefore extremely relevant. Here, we describe the development of the Capsicum pepper bioinspired peptide “capsicumicine.” By using microbiological, microscopic, and nuclear magnetic resonance (NMR) approaches, we demonstrate that capsicumicine strongly prevents methicillin-resistant Staphylococcus epidermidis biofilm via an extracellular “matrix anti-assembly” mechanism of action. The results were confirmed in vivo in a translational preclinical model that mimics medical device-related infection. Since capsicumicine is not cytotoxic, it is a promising candidate for complementary treatment of infectious diseases

Reflux of Endoplasmic Reticulum proteins to the cytosol inactivates tumor suppressors

International audienceIn the past decades, many studies reported the presence of endoplasmic reticulum (ER)-resident proteins in the cytosol. However, the mechanisms by which these proteins relocate and whether they exert cytosolic functions remain unknown. We find that a subset of ER luminal proteins accumulates in the cytosol of glioblastoma cells isolated from mouse and human tumors. In cultured cells, ER protein reflux to the cytosol occurs upon ER proteostasis perturbation. Using the ER luminal protein anterior gradient 2 (AGR2) as a proof of concept, we tested whether the refluxed proteins gain new functions in the cytosol. We find that refluxed, cytosolic AGR2 binds and inhibits the tumor suppressor p53. These data suggest that ER reflux constitutes an ER surveillance mechanism to relieve the ER from its contents upon stress, providing a selective advantage to tumor cells through gain-of-cytosolic functions-a phenomenon we name ER to Cytosol Signaling (ERCYS)

Plateau de microscopie électronique à transmission

National audiencePrésentation des offres de service, des équipements disponibles et des techniques mises en oeuvre sur le plateau de ME

Milk Secretion: The Role of SNARE Proteins: Milk Secretion: The Role of SNARE Proteins

International audienceDuring lactation, polarized mammary epithelial secretory cells (MESCs) secrete huge quantities of the nutrient molecules that make up milk, i.e. proteins, fat globules and soluble components such as lactose and minerals. Some of these nutrients are only produced by the MESCs themselves, while others are to a great extent transferred from the blood. MESCs can thus be seen as a crossroads for both the uptake and the secretion with cross-talks between intracellular compartments that enable spatial and temporal coordination of the secretion of the milk constituents. Although the physiology of lactation is well understood, the molecular mechanisms underlying the secretion of milk components remain incompletely characterized. Major milk proteins, namely caseins, are secreted by exocytosis, while the milk fat globules are released by budding, being enwrapped by the apical plasma membrane. Prolactin, which stimulates the transcription of casein genes, also induces the production of arachidonic acid, leading to accelerated casein transport and/or secretion. Because of their ability to form complexes that bridge two membranes and promote their fusion, SNARE (Soluble N-ethylmaleimide-Sensitive Factor Attachment Protein Receptor) proteins are involved in almost all intracellular trafficking steps and exocytosis. As SNAREs can bind arachidonic acid, they could be the effectors of the secretagogue effect of prolactin in MESCs. Indeed, some SNAREs have been observed between secretory vesicles and lipid droplets suggesting that these proteins could not only orchestrate the intracellular trafficking of milk components but also act as key regulators for both the coupling and coordination of milk product secretion in response to hormones

Reticulate evolution in kiwifruit (Actinidia, Actinidiaceae) identified by comparing their maternal and paternal phylogenies

Evolutionary relationships within Actinidia, a genus known for the contrasting mode of inheritance of its plastids and mitochondria, were studied. The phylogenetic analysis is based on chloroplast (cp) and mitochondrial (mt) restriction site and sequence data (matK, psbC-trnS, rbcL, and trnL-trnF for cpDNA; nad1-2/3 and nad4-1/2 for mtDNA). The analysis of cp sequence data confirms the hypothesis that the four currently recognized sections are not monophyletic. The detection of incongruences among phylogenies (mtDNA vs. cpDNA tree) coupled with the detection of intraspecific polymorphisms confirms some of the reticulations previously emphasized, diagnoses new hybridization/introgression events, and provides evidence for multiple origin of at least two polyploid taxa. A number of hybridization/introgression events at the diploid, tetraploid, and possibly hexaploid levels are documented. The extensive reticulate evolution undergone by Actinidia could account for the lack of clear morphological discontinuities at the species leve

Antigen recovery and preservation using the microwave irradiation of biological samples for transmission electron microscopy analysis

International audienceMost studies using microwave irradiation (MWI) for the preparation of tissue samples have reported an improvement in structural integrity. However, there have been few studies on the effect of microwave (MW) on antigen preservation during sample preparation prior to immunolocalization. This report documents our experience of specimen preparation using an automatic microwave apparatus to obtain antigen preservation and retrieval. We tested the effects of MW processing vs. conventional procedures on the morphology and antigenicity of two different tissues: the brain and mammary gland, whose chemical composition and anatomical organization are quite different. We chose to locate the transcription factor PPARβ/δ using immunocytochemistry on brain tissue sections from hamsters. Antigen retrieval protocols involving MWI were used to restore immunoreactivity. We also studied the efficiency of the ultrastructural immunolocalization of both PPARγ and caveolin-1 following MWI vs. conventional treatment, on mammary gland tissue from mice at 10 days of lactation. Our findings showed that the treatment of tissue samples with MWI, in the context of a process lasting just a few hours from fixation to immunolocalization, enabled similar, or even better, results than conventional protocols. The quantification of immunolabeling for cav-1 indicated an increase in density of up to three-fold in tissues processed in the microwave oven. Furthermore, MW treatment permitted the localization of PPARβ/δ in glutaraldehyde-fixed specimens, which was impossible in the absence of MWI. This study thus showed that techniques involving the use of microwaves could largely improve both ultrastructure and immunodetection

Le plateau de microscopie électronique à transmission de l'Inra de Jouy-en-Josas

National audienceLe plateau technique de microscopie électronique à transmission est un des trois plateaux d'imagerie qui constituent la plateforme d'imagerie MIMA2 (Microscopie et Imagerie des Micro-organismes, Animaux et Aliments). La plate-forme est par ailleurs équipée d'appareils dédiés à la microscopie optique (confocal et apotome) et à la microscopie électronique à balayage. Cette structure est ouverte aux équipes extérieures au site. Les projets développés en interne sur cette plate-forme concernent avant tout l'animal et les procaryotes (bactéries). Les personnels du plateau offre la possibilité d'apprendre la préparation des échantillons et des formations sur les techniques d'observation en microscopie électronique. Ces techniques peuvent être appliquées et adaptées dans les différentes unités de recherche à divers projets et objets pour une observation à l'échelle 28 subcellulaire. Le plateau met en place des approches de microscopie corrélative pour des questions de biologie cellulaire et du développement. Des approches d’analyse élémentaires (EELS et PEELS) sont également réalisées au sein du plateau de microscopie sur un microscope ZEISS EM902, équipé d’un filtre alpha de perte d’énergie. La structure, l'activité et quelques exemples d'études réalisées par le plateau technique sont décrits

Structural insights into the binding of bS1 to the ribosome

International audienceThe multidomain ribosomal protein bS1 is the biggest and the most flexible and dynamic protein in the 30S small subunit. Despite being essential for mRNA recruitment and its primary role in the accommodation of the start codon within the decoding centre, there has not yet been a high-resolution description of its structure. Here, we present a 3D atomic model of OB1 and OB2, bS1's first two N-terminal domains, bound to an elongation-competent 70S ribosome. Our structure reveals that, as previously reported, bS1 is anchored both by a pi-stacking to the 30S subunit and via a salt bridge with the Zn2+ pocket of bS1. These contacts are further stabilized by other interactions with additional residues on OB1. Our model also shows a new conformation of OB2, interacting with the Shine-Dalgarno portion of the mRNA. This study confirms that OB1 plays an anchoring role, but also highlights a novel function for OB2, which is directly involved in the modulation and support of mRNA binding and accommodation on the ribosome