Etude comparée de la régulation par le calcium de l'adressage de l'aquaporine-3 et- de l'aquaporine-2 dans les cellules épithéliales.

Les aquaporines (AQPs) sont de petites protéines membranaires permettant le passage facilité de l eau, du glycérol et de certains solutés à travers les membranes biologiques. Elles jouent d importants rôles de transport transmembranaires ou transcellulaires dans diverses cellules telles que les cellules rénales, mais aussi dans les kératinocytes de l'épiderme. L épiderme est un épithélium pluristratifié en constant renouvellement. Le calcium extracellulaire joue un rôle important dans le mécanisme de différenciation des kératinocytes.Dans ce travail, nous avons montré que la différenciation induite par le calcium de kératinocytes humains s'accompagne de l adressage de l aquaporine-3 (AQP3) du réticulum endoplasmique, vers les membranes plasmiques. Pour étudier la cinétique et les bases moléculaires de cette régulation, notre objectif était de produire des clones stables d'une lignée de kératinocytes humains en culture (HaCat) exprimant une AQP3 fluorescente. Malgré plusieurs tentatives, je n'ai pas pu obtenir ces clones stables. J'ai alors choisi un autre modèle de cellules épithéliales en culture; les cellules MDCK. Nous avons produit deux lignées stables de MDCK exprimant des aquaporines fluorescentes: l'AQP3-GFP et l'AQP2-mCherry. De manière intéressante, dans les cellules MDCK, l'AQP3 -GFP reproduit la régulation de son adressage par le calcium observée dans les kératinocytes humains; dans des cellules MDCK cultivées en présence de 0,15mM de Ca2+, l AQP3-GFP est localisée dans le réticulum endoplasmique, tandis qu à 1,5mM de Ca2+ extracellulaire, celle-ci est localisée aux membranes plasmiques. Dans les mêmes conditions, l'AQP2-mCherry conserve une localisation intracellulaire. Par des expériences de calcium switch , nous avons étudié la cinétique du trafic cellulaire de l'AQP3 et montré que l'adressage de l AQP3 à la membrane plasmique en réponse au calcium est lent (6h minimum) et semble dépendant non seulement de la différenciation cellulaire, mais aussi de l'établissement de la polarité cellulaire. A l aide d inhibiteurs de la PLC et de la PKC, nous avons montré l implication de cette voie de signalisation, qui dépend du calcium, dans le trafic de l AQP3. De plus, l'adressage membranaire de l'AQP3 est dépendant du cytosquelette d actine.En conclusion, nous montrons pour la première fois une régulation du trafic intracellulaire d'une aquaporine par le calcium au cours de la différenciation et de l'établissement de la polarité cellulaire de cellules épithéliales. Cette régulation permet probablement l'hydratation de l'épiderme humain, sans remettre en cause la barrière de perméabilité que constitue la peau.The aquaporins (AQPs) are small membrane proteins forming water channels and transporters for smal solutes like glycerol. The AQPs play important roles in transmembrane or transcellular transports in various cells, like kidney cells, but also in skin epidermis keratinocytes. The skin epidermis is a pluristratified epithelium, undergoing continuous renewal. Extracellular calcium plays an important role in the differentiation of keratinocytes.In this work, we demonstrate that during calcium-induced differentiation of human keratinocytes, aquaporin-3 (AQP3) is translocated from the endoplasmic reticulum to plasma membranes. In order to study the kinetics and the molecular bases of this regulation, our goal was to produce stable clones of a human keratinocyte cell line (HaCat) expressing a fluorescent AQP3. Despite several trials, i was not able to obtain such clones. Thus i pursued with another epithelial cell line: MDCK cells. We have produced two lines of MDCK cells stably expressing fluorescent AQPs: AQP3-GFP and AQP2mCherry. Interestingly in MDCK cells, AQP3-GFP reproduced the regulated intracellular trafficking observed in human keratinocytes; in MDCK cells grown in a medium containing 0.15 mM Ca2+,, AQP3-GFP was localized in the endoplasmic reticulum. After extracellular Ca2+ was raised to 1.5 mM, AQP3-GFP was seen in plasma membranes. In the same conditions, AQP2-mCherry remained intracellular throughtout the experiment. With calcium-switch experiments, when have then studied the kinetics of AQP3 trafficking. We have shown that targeting of AQP3 to plasma membranes is a slow process (at least 6h) and seems dependent not only of cell differentiation, but also on the establishment of cell polarity. Using inhibitors of PLC and PKC, we have shown the implication of this signalling pathway, which is dependent on calcium, in AQP3 trafficking. In addition we found that plasma membrane expression of AQP3 is dependent on actin cytoskeleton.In conclusion, we show for the first time a regulation of intracelluar trafficking of an aquaporin in calcium-induced differentiation and after establishment of epithelial cell polarity. This regulation likely allows human skin epidermis hydration whithout compromising the permeability barrier of skin.PARIS11-SCD-Bib. électronique (914719901) / SudocSudocFranceF

Optimisation of recombinant production of active human cardiac SERCA2a ATPase

Methods for recombinant production of eukaryotic membrane proteins, yielding sufficient quantity and quality of protein for structural biology, remain a challenge. We describe here, expression and purification optimisation of the human SERCA2a cardiac isoform of Ca2+ translocating ATPase, using Saccharomyces cerevisiae as the heterologous expression system of choice. Two different expression vectors were utilised, allowing expression of C-terminal fusion proteins with a biotinylation domain or a GFP- His8 tag. Solubilised membrane fractions containing the protein of interest were purified onto Streptavidin-Sepharose, Ni-NTA or Talon resin, depending on the fusion tag present. Biotinylated protein was detected using specific antibody directed against SERCA2 and, advantageously, GFP-His8 fusion protein was easily traced during the purification steps using in-gel fluorescence. Importantly, talon resin affinity purification proved more specific than Ni-NTA resin for the GFP-His8 tagged protein, providing better separation of oligomers present, during size exclusion chromatography. The optimised method for expression and purification of human cardiac SERCA2a reported herein, yields purified protein (> 90%) that displays a calcium-dependent thapsigargin-sensitive activity and is suitable for further biophysical, structural and physiological studies. This work provides support for the use of Saccharomyces cerevisiae as a suitable expression system for recombinant production of multi-domain eukaryotic membrane proteins

mRNP3 and mRNP4 are phosphorylatable by casein kinase II in Xenopus oocytes, but phosphorylation does not modify RNA-binding affinity

AbstractmRNP3 and mRNP4 (also called FRGY2) are two mRNA-binding proteins which are major constituents of the maternal RNA storage particles of Xenopus laevis oocytes. The phosphorylation of mRNP3–4 has been implicated in the regulation of mRNA masking. In this study, we have investigated their phosphorylation by casein kinase II and its consequence on their affinity for RNA. Comparison of the phosphopeptide map of mRNP3–4 phosphorylated in vivo with that obtained after phosphorylation in vitro by purified Xenopus laevis casein kinase II strongly suggests that casein kinase II is responsible for the in vivo phosphorylation of mRNP3–4 in oocytes. The phosphorylation occurs on a serine residue in a central domain of the proteins. The affinity of mRNP3–4 for RNA substrates remained unchanged after the treatment with casein kinase II or calf intestine phosphatase in vitro. This suggests that phosphorylation of these proteins does not regulate their interaction with RNA but rather controls their interactions with other proteins

Modelling the hydrological behaviour of a coffee agroforestry basin in Costa Rica

UMR LISAH, Equipe Eau et Polluants en Bassins VersantsThe profitability of hydropower in Costa Rica is affected by soil erosion and sedimentation in dam reservoirs, which are in turn influenced by land use, infiltration and aquifer interactions with surface water. In order to foster the provision and payment for Hydrological Environmental Services (HES), a quantitative assessment of the impact of specific land uses on the functioning of drainage-basins is required. The present paper aims to study the water balance partitioning in a volcanic coffee agroforestry microbasin (1 km(2), steep slopes) in Costa Rica, as a first step towards evaluating sediment or contaminant loads. The main hydrological processes were monitored during one year, using flume, eddy-covariance flux tower, soil water profiles and piezometers. A new Hydro-SVAT lumped model is proposed, that balances SVAT (Soil Vegetation Atmosphere Transfer) and basin-reservoir routines. The purpose of such a coupling was to achieve a trade-off between the expected performance of ecophysiological and hydrological models, which are often employed separately and at different spatial scales, either the plot or the basin. The calibration of the model to perform streamflow yielded a Nash-Sutcliffe (NS) coefficient equal to 0.89 for the year 2009, while the validation of the water balance partitioning was consistent with the independent measurements of actual evapotranspiration (R-2 = 0.79, energy balance closed independently), soil water content (R-2 = 0.35) and water table level (R-2 = 0.84). Eight months of data from 2010 were used to validate modelled streamflow, resulting in a NS = 0.75. An uncertainty analysis showed that the streamflow modelling was precise for nearly every time step, while a sensitivity analysis revealed which parameters mostly affected model precision, depending on the season. It was observed that 64% of the incident rainfall R flowed out of the basin as streamflow and 25% as evapotranspiration, while the remaining 11% is probably explained by deep percolation, measurement errors and/or inter-annual changes in soil and aquifer water stocks. The model indicated an interception loss equal to 4% of R, a surface runoff of 4% and an infiltration component of 92%. The modelled streamflow was constituted by 87% of baseflow originating from the aquifer, 7% of subsurface non-saturated runoff and 6% of surface runoff. Given the low surface runoff observed under the current physical conditions (andisol) and management practices (no tillage, planted trees, bare soil kept by weeding), this agroforestry system on a volcanic soil demonstrated potential to provide valuable HES, such as a reduced superficial displacement- capacity for fertilizers, pesticides and sediments, as well as a streamflow regulation function provided by the highly efficient mechanisms of aquifer recharge and discharge. The proposed combination of experimentation and modelling across ecophysiological and hydrological approaches proved to be useful to account for the behaviour of a given basin, so that it can be applied to compare HES provision for different regions or management alternatives

Serca1 Truncated Proteins Unable to Pump Calcium Reduce the Endoplasmic Reticulum Calcium Concentration and Induce Apoptosis

By pumping calcium from the cytosol to the ER, sarco/endoplasmic reticulum calcium ATPases (SERCAs) play a major role in the control of calcium signaling. We describe two SERCA1 splice variants (S1Ts) characterized by exon 4 and/or exon 11 splicing, encoding COOH terminally truncated proteins, having only one of the seven calcium-binding residues, and thus unable to pump calcium. As shown by semiquantitative RT-PCR, S1T transcripts are differentially expressed in several adult and fetal human tissues, but not in skeletal muscle and heart. S1T proteins expression was detected by Western blot in nontransfected cell lines. In transiently transfected cells, S1T homodimers were revealed by Western blot using mildly denaturing conditions. S1T proteins were shown, by confocal scanning microscopy, to colocalize with endogenous SERCA2b into the ER membrane. Using ER-targeted aequorin (erAEQ), we have found that S1T proteins reduce ER calcium and reverse elevation of ER calcium loading induced by SERCA1 and SERCA2b. Our results also show that SERCA1 variants increase ER calcium leakage and are consistent with the hypothesis of a cation channel formed by S1T homodimers. Finally, when overexpressed in liver-derived cells, S1T proteins significantly induce apoptosis. These data reveal a further mechanism modulating Ca2+ accumulation into the ER of nonmuscle cells and highlight the relevance of S1T proteins to the control of apoptosis