Regulation of exocytosis in the mammary secreotry cell, and the role of SNARE proteins

National audienceDuring lactation, mammary epithelial cells (MECs) secrete a huge amount of milk, an aqueous fluid containing proteins, milk fat globules (MFGs) and elements such as lactose and minerals. These nutrients have two origins: some are produced by the MEC, while others are transferred from blood to milk by transcytosis. The MEC can thus be seen as a crossroad for both the uptake and the vectorial secretion of the milk constituents. These processes are likely to involve a cross-talk between the endocytic/exocytic compartments in order to regulate spatio-temporally the secretion of milk products. Although the physiology of lactation is well understood, the molecular mechanisms underlying the secretion of milk products are still poorly characterized. The major milk proteins, namely caseins, are secreted by exocytosis while the MFGs are released by budding at the apical plasma membrane. Casein exocytosis thus provides membrane which may be reused to enwrap the MFG during its budding. Although milk secretion appears to be mostly constitutive, prolactin was shown to activate a phospholipase A2 which, in turn, produces arachidonic acid, leading to the acceleration of casein transport and/or secretion. Thus, MECs may possess both the constitutive and the regulated secretory pathways. Whatever their secretory mode, however, intracellular trafficking and exocytosis of the caseins probably involve SNARE (Soluble NSF Attachment Protein (SNAP) Receptor) proteins. Due to their ability to form highly stable four-helix bundle complex bridging a donor and an acceptor membranes, SNAREs promote membrane fusion in a targeted manner. Moreover, SNAREs bind arachidonic acid, thus facilitating exocytosis. In MECs, certain SNAREs are associated with both casein-containing vesicles and intracellular lipid droplets. By orchestrating the intracellular trafficking of milk components in a hormonally responsive manner, SNAREs may therefore contribute as a key point for the regulation of both the coupling and the coordination of milk product secretion at time of suckling

Indirect immunofluorescence on frozen sections of mouse mammary gland

Indirect immunofluorescence is used to detect and locate proteins of interest in a tissue. The protocol presented here describes a complete and simple method for the immune detection of proteins, the mouse lactating mammary gland being taken as an example. A protocol for the preparation of the tissue samples, especially concerning the dissection of mouse mammary gland, tissue fixation and frozen tissue sectioning, are detailed. A standard protocol to perform indirect immunofluorescence, including an optional antigen retrieval step, is also presented. The observation of the labeled tissue sections as well as image acquisition and post-treatments are also stated. This procedure gives a full overview, from the collection of animal tissue to the cellular localization of a protein. Although this general method can be applied to other tissue samples, it should be adapted to each tissue/primary antibody couple studied

Mammary gland secretion: hormonal coordination of endocytosis and exocytosis

International audienceThe mammary epithelium coordinates the uptake of milk precursors and the transport of milk components in order to produce milk of relatively constant composition at a particular stage of lactation, as long as the mammary gland is healthy. The mammary epithelial cell controls the uptake of blood-borne molecules at its basal side and the release of products into milk at its apical side, through mechanisms of internalization (endocytosis) and mechanisms of release (exocytosis). These events are strictly dependent on the physiological stage of the mammary gland. This review addresses the mechanisms responsible for these processes and points out new questions that remain to be answered concerning possible interconnections between them, for an optimal milk secretion

Voies de signalisation de l'interféron-gamma dans les ovocytes et les embryons pré-implantatoires de souris

PARIS-Museum Hist.Naturelle (751052304) / SudocSudocFranceF

Mouse oocytes and preimplantation embryos bear the two sub-units of interferon-gamma receptor

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Matrix metalloproteinase-9 silencing by RNA interference triggers the migratory-adhesive switch in Ewing’s sarcoma cells : MMP-9 silencing alters Ewing’s sarcoma motility

Enhanced expression of (pro)matrix metalloproteinase-9 (MMP-9) is associated with human tumor invasion and/or metastasis. COH cells derived from a highly invasive and metastatic Ewing’s sarcoma constitutively express proMMP-9. Transfection of a double stranded RNA that targets the MMP-9 mRNA into COH cells depleted the corresponding mRNA and protein as demonstrated by reverse transcriptase-PCR, enzymelinked immunosorbent assay, and gelatin zymography. proMMP-9 extinction resulted in the following: (i) decreased spreading on extracellular matrix (fibronectin, laminin, collagen IV)-coated surfaces, (ii) inhibition of migration toward fibronectin, and (iii) induced aggregation, which was specifically disrupted by a functionblocking E-cadherin antibody. MMP-9 knockdown concomitantly resulted in increased levels of surface E-cadherin, redistribution at the plasma membrane of beta-catenin, and its physical association with E-cadherin. Moreover, induction of E-cadherin-mediated adhesion was associated with RhoA activation and changes in paxillin cytoskeleton. Finally, an inhibitor of gelatinolytic activity of pro-MMP9 did not reduce COH cell migration confirming that the enzymatic property of COH MMP-9 was not required for migration toward fibronectin. Overall, our observations define a novel critical role for proMMP-9 in providing a cellular switch between stationary and migratory cell phases

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

Molecular mechanisms of caseins exocytosis: role of the SNARE proteins

National audienceThe mammary gland (MG) is a complex tissue which function is to ensure the offspring’s feeding by producing and secreting the milk components during lactation. In addition to stimulate the synthesis of the main milk proteins, the caseins, prolactin (PRL) also induces an increase of arachidonic acid (AA) in mammary epithelial cells (MECs). This lipid second messenger may accelerate the transport and possibly the exocytosis of caseins (secretagogue effect of PRL, [1]). SNARE (Soluble NSF Attachment Receptor) proteins are able to form stable ternary complexes (SNARE complexes) which promote the fusion of two membranous compartments [2, 3]. Indeed, SNAREs are involved in intracellular traffic and exocytosis processes in many cell types. SNAREs have recently been described as target for AA, thus promoting exocytosis in neuroendocrine cells [4, 5]. Milk products secretion is known in general terms but the molecular mechanisms underlying caseins secretion have not been described to date. One intriguing hypothesis is that the AA produced in response to PRL may regulate the transport and the exocytosis of caseins by targeting the SNARE proteins in MECs during lactation. We show for the first time that mRNAs encoding most of the SNARE proteins are present in the mouse MG and MECs at different developmental stages. Moreover, quantitative Western blots show that levels of some SNARE proteins are regulated during the development of the MG. The localization of the SNARE proteins was investigated in lactating mouse MG by indirect immunofluorescence and electron microscopy. We found that both the developmental stage of the MG and the suckling induce important changes in the localization of certain SNARE proteins. The association of some SNARE proteins with the lipid droplets and the vesicles containing caseins was observed in lactating MECs. We also noticed the presence of gold particles corresponding to SNAP-23 at the interface between the lipid droplets and the caseins vesicles membranes. Immunoprecipitation experiments were performed to identify the SNARE complex involved in caseins exocytosis in murine MECs. The identification of the proteins engaged in SNARE complexes was investigated by Western Blot and mass spectrometry. The expression of some regulatory proteins of the SNARE complex such as synaptotagmins, Munc13 and 18, Rab3, NSF and alpha-SNAP was also investigated in murine MECs by RT-PCR. [1] Ollivier-Bousquet et al. J Nutr, 1993. 123(12):2090-100. [2] Rothman & Orci. Nature, 1992. 355(6359):409-15. [3] Sollner et al. Nature, 1993. 362(6418):318-24. [4] Latham et al. J Neuroche, 2007. 100(6):1543-54. [5] Darios & Davletov. Nature, 2006. 440(7085):813-7

Expression des microARN pendant le développement de la glande mammaire ovine adulte

National audienceIntroduction : La glande mammaire subit de nombreux remodelages durant la gestation et la lactation mettant en œuvre de nombreux processus cellulaires (i.e. prolifération et différenciation cellulaire, apoptose...). Ces processus sont sous le contrôle de nombreux types de régulateurs, notamment les microARN (miRNA). Résultats: Après construction d’une banque de miRNA dans la glande mammaire ovine en début de gestation, nous avons cloné 54 nouveaux miRNA jamais décrits chez le mouton. Les séquences de ces miRNA sont très conservées par rapport à leur équivalent chez la vache, la souris ou l’homme. Ces résultats ont été complétés par une étude suivant l’expression des miRNA pendant le développement de la glande mammaire ovine adulte, en utilisant la technique de microarray. Nous avons montré qu’une centaine de miRNA est régulée suivant trois principaux profils d’expression: une baisse d’expression pendant la gestation et la lactation, un pic d’expression pendant la gestation ou une augmentation progressive d’expression à partir de la fin de gestation et au cours de la lactation. L’accumulation d’un miRNA représentatif de chacun de ces profils (respectivement miR-21, miR-205 et miR-200b) a été suivie par hybridation in situ à plusieurs stades du développement de la glande mammaire ovine. MiR-21 et miR-200b ont été détectés dans les cellules épithéliales luminales, alors que miR-205, quant à lui, l’a été dans des cellules basales pendant la première moitié de la gestation puis dans les cellules luminales pendant la deuxième moitié. Conclusion: Nos résultats décrivent une forte expression de miR-21 dans la glande mammaire pendant la gestation précoce, période correspondant à une prolifération cellulaire intense. Par ailleurs, nous montrons que miR-205 commence à être exprimé dans les cellules luminales pendant la seconde moitié de la gestation, alors que miR-200b est déjà présent dans ces cellules. Ces deux miRNA pourraient ainsi coopérer, comme cela a déjà été décrit dans une lignée cellulaire épithéliale (MDCK), notamment afin de maintenir le statut épithélial de ces cellules en réprimant un programme comparable à l’EMT (transition epithélio-mésenchymateuse) de façon à atteindre puis préserver le phénotype sécrétoire des cellules épithéliales mammaires. Perspectives : Afin de déterminer l’implication de ces miRNA dans ces régulations, nous envisageons de bloquer l’action de ces miRNA dans un modèle de dédifférenciation cellulaire (acini mammaires de souris en culture in vitro), puis de suivre la morphologie de ces cellules ainsi que l’expression de marqueurs épithéliaux et mésenchymaux