Anne Cantereau

Syntaxin 8 impairs trafficking of cystic fibrosis transmembrane conductance regulator (CFTR) and inhibits its channel activity

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMP-dependent chloride channel that mediates electrolyte transport across the luminal surface of epithelial cells. In this paper, we describe the CFTR regulation by syntaxin 8, a t-SNARE protein (target soluble N-ethylmaleimide-sensitive factor attachment protein receptor) involved in the SNARE endosomal complex. Syntaxin family members are key molecules implicated in diverse vesicle docking and membrane fusion events. We found that syntaxin 8 physically interacts with CFTR: recombinant syntaxin 8 binds CFTR in vitro and both proteins co-immunoprecipitate in HT29 cells. Syntaxin 8 regulates CFTR-mediated currents in chinese hamster ovary (CHO) cells stably expressing CFTR and syntaxin 8. Iodide efflux and whole-cell patch-clamp experiments on these cells indicate a strong inhibition of CFTR chloride current by syntaxin 8 overexpression. At the cellular level, we observed that syntaxin 8 overexpression disturbs CFTR trafficking. Confocal microscopy shows a dramatic decrease in green fluorescent protein-tagged CFTR plasma membrane staining, when syntaxin 8 is coexpressed in COS-7 cells. Using antibodies against Lamp-1, TfR or Rab11 we determined by immunofluorescence assays that both proteins are mainly accumulated in recycling endosomes. Our results evidence that syntaxin 8 contributes to the regulation of CFTR trafficking and chloride channel activity by the SNARE machinery.

Interaction of grape ASR proteins with a DREB transcription factor in the nucleus.

MINT‐6743067: VvMSA (uniprotkb:Q94G23) and VvDREB (uniprotkb:A6XA90) physically interact (MI:0218) by bimolecular fluorescence complementation (MI:0809) MINT‐6743043: VvMSA (uniprotkb:Q94G23) physically interacts (MI:0218) with VvDREB (uniprotkb:A6XA90) by two hybrid (MI:0018)

Molecular and functional characterization of a new potassium conductance in mouse ventricular fibroblasts

The present work is aimed at identifying and characterizing, at a molecular and functional level, new ionic conductances potentially involved in the excitation-secretion coupling and proliferation of cardiac ventricular fibroblasts. Among potassium channel transcripts which were screened by high-throughput real-time PCR, SUR2 and Kir6.1 mRNAs were found to be the most abundant in ventricular fibroblasts. The corresponding proteins were not detected by western blot following 5 days of cell culture, but had appeared at 7 days, increasing with extended cell culture duration as the fibroblasts differentiated into myofibroblasts. Using the inside-out configuration of the patch-clamp technique, single potassium channels could be recorded. These had properties similar to those reported for SUR2/Kir6.1 channels, i.e. activation by pinacidil, inhibition by glibenclamide and activation by intracellular UDP. As already reported for this molecular signature, they were insensitive to intracellular ATP. In the whole-cell configuration, these channels have been shown to be responsible for a glibenclamide-sensitive macroscopic potassium current which can be activated not only by pinacidil, but also by nanomolar concentrations of the sphingolipid sphingosine-1-phosphate (S1P). The activation of this current resulted in an increase in cell proliferation and a decrease in IL-6 secretion, suggesting it has a functional role in situations where S1P increases. Overall, this work demonstrates for the first time that SUR2/Kir6.1 channels represent a significant potassium conductance in ventricular fibroblasts which may be activated in physio-pathological conditions and which may impact on fibroblast proliferation and function.

Na+/Ca2+ exchange in human myotubes: intracellular calcium rises in response to external sodium depletion are enhanced in DMD

This study aims to investigate the sodium/calcium exchanger expression in human co-cultured skeletal muscle cells and to compare the effects of Na(+)/Ca(2+) exchange activity in normal and dystrophic (Duchennes muscular dystrophy) human co-cultured myotubes. For this purpose, variations of intracellular calcium concentration ([Ca(2+)](int)) were monitored, as the variations of the fluorescence ratio of indo-1 probe, in response to external sodium depletion. External sodium withdrawal induced [Ca(2+)](int) rises within several seconds in both normal and Duchennes muscular dystrophy myotubes. These Na(+)-free-induced [Ca(2+)](int) elevations were attributed to the reverse mode of the Na(+)/Ca(2+) exchange mechanism since the phenomenon was dependent on extracellular calcium concentration ([Ca(2+)](ext)), and since it was sensitive to external Ni(2+) ions. Amplitudes of Na(+)-free-induced [Ca(2+)](int) rises were significantly greater in Duchennes muscular dystrophy cells than in normal ones. Such a difference disappeared when the sarcoplasmic reticulum was pharmacologically blocked, suggesting that the reverse mode of the Na(+)/Ca(2+) exchange mechanism was able to generate enhanced calcium-induced calcium-release in Duchennes muscular dystrophy myotubes. Immunostaining images of Na(+)/Ca(2+) exchanger (NCX) isoforms, obtained by confocal microscopy, revealed the presence of NCX1 and NCX3 at the sarcolemmal level of both normal and Duchennes muscular dystrophy myotubes. No differences were observed in the location of NCX isoforms expression between normal and Duchennes muscular dystrophy co-cultured myotubes.

Abnormal spatial diffusion of Ca2+ in F508del-CFTR airway epithelial cells

BackgroundIn airway epithelial cells, calcium mobilization can be elicited by selective autocrine and/or paracrine activation of apical or basolateral membrane heterotrimeric G protein-coupled receptors linked to phospholipase C (PLC) stimulation, which generates inositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol (DAG) and induces Ca2+ release from endoplasmic reticulum (ER) stores.MethodsIn the present study, we monitored the cytosolic Ca2+ transients using the UV light photolysis technique to uncage caged Ca2+ or caged IP3 into the cytosol of loaded airway epithelial cells of cystic fibrosis (CF) and non-CF origin. We compared in these cells the types of Ca2+ receptors present in the ER, and measured their Ca2+ dependent activity before and after correction of F508del-CFTR abnormal trafficking either by low temperature or by the pharmacological corrector miglustat (N-butyldeoxynojirimycin).ResultsWe showed reduction of the inositol 1,4,5-trisphosphate receptors (IP3R) dependent-Ca2+ response following both correcting treatments compared to uncorrected cells in such a way that Ca2+ responses (CF+treatment vs wild-type cells) were normalized. This normalization of the Ca2+ rate does not affect the activity of Ca2+-dependent chloride channel in miglustat-treated CF cells. Using two inhibitors of IP3R1, we observed a decrease of the implication of IP3R1 in the Ca2+ response in CF corrected cells. We observed a similar Ca2+ mobilization between CF-KM4 cells and CFTR-cDNA transfected CF cells (CF-KM4-reverted). When we restored the F508del-CFTR trafficking in CFTR-reverted cells, the specific IP3R activity was also reduced to a similar level as in non CF cells. At the structural level, the ER morphology of CF cells was highly condensed around the nucleus while in non CF cells or corrected CF cells the ER was extended at the totality of cell.ConclusionThese results suggest reversal of the IP3R dysfunction in F508del-CFTR epithelial cells by correction of the abnormal trafficking of F508del-CFTR in cystic fibrosis cells. Moreover, using CFTR cDNA-transfected CF cells, we demonstrated that abnormal increase of IP3R Ca2+ release in CF human epithelial cells could be the consequence of F508del-CFTR retention in ER compartment.

A chimerical phagocytosis model reveals the recruitment by Sertoli cells of autophagy for the degradation of ingested illegitimate substrates.

Phagocytosis and autophagy are typically dedicated to degradation of substrates of extrinsic and intrinsic origins respectively. Although overlaps between phagocytosis and autophagy were reported, the use of autophagy for ingested substrate degradation by nonprofessional phagocytes has not been described. Blood-separated tissues use their tissue-specific nonprofessional phagocytes for homeostatic phagocytosis. In the testis, Sertoli cells phagocytose spermatid residual bodies produced during germ cell differentiation. In the retina, pigmented epithelium phagocytoses shed photoreceptor tips produced during photoreceptor renewal. Spermatid residual bodies and shed photoreceptor tips are phosphatidylserine-exposing substrates. Activation of the tyrosine kinase receptor MERTK, which is implicated in phagocytosis of phosphatidylserine-exposing substrates, is a common feature of Sertoli and retinal pigmented epithelial cell phagocytosis. The major aim of our study was to investigate to what extent phagocytosis by Sertoli cells may be tissue specific. We analyzed in Sertoli cell cultures that were exposed to either spermatid residual bodies (legitimate substrates) or retina photoreceptor outer segments (illegitimate substrates) the course of the main phagocytosis stages. We show that whereas substrate binding and ingestion stages occur similarly for legitimate or illegitimate substrates, the degradation of illegitimate but not of legitimate substrates triggers autophagy as evidenced by the formation of double-membrane wrapping, MAP1LC3A-II/LC3-II clustering, SQSTM1/p62 degradation, and by marked changes in ATG5, ATG9 and BECN1/Beclin 1 protein expression profiles. The recruitment by nonprofessional phagocytes of autophagy for the degradation of ingested cell-derived substrates is a novel feature that may be of major importance for fundamentals of both apoptotic substrate clearance and tissue homeostasis.

Mini-dystrophin expression down-regulates IP3-mediated calcium release events in resting dystrophin-deficient muscle cells.

We present here evidence for the enhancement, at rest, of an inositol 1,4,5-trisphosphate (IP3)–mediated calcium signaling pathway in myotubes from dystrophin-deficient cell lines (SolC1(−)) as compared to a cell line from the same origin but transfected with mini-dystrophin (SolD(+)). With confocal microscopy, the number of sites discharging calcium (release site density [RSD]) was quantified and found more elevated in SolC1(−) than in SolD(+) myotubes. Variations of membrane potential had no significant effect on this difference, and higher resting [Ca2+]i in SolC1(−) (Marchand, E., B. Constantin, H. Balghi, M.C. Claudepierre, A. Cantereau, C. Magaud, A. Mouzou, G. Raymond, S. Braun, and C. Cognard. 2004. Exp. Cell Res. 297:363–379) cannot explain alone higher RSD. The exposure with SR Ca2+ channel inhibitors (ryanodine and 2-APB) and phospholipase C inhibitor (U73122) significantly reduced RSD in both cell types but with a stronger effect in dystrophin-deficient SolC1(−) myotubes. Immunocytochemistry allowed us to localize ryanodine receptors (RyRs) as well as IP3 receptors (IP3Rs), IP3R-1 and IP3R-2 isoforms, indicating the presence of both RyRs-dependent and IP3-dependent release systems in both cells. We previously reported evidence for the enhancement, through a Gi protein, of the IP3-mediated calcium signaling pathway in SolC1(−) as compared to SolD(+) myotubes during a high K+ stimulation (Balghi, H., S. Sebille, B. Constantin, S. Patri, V. Thoreau, L. Mondin, E. Mok, A. Kitzis, G. Raymond, and C. Cognard. 2006. J. Gen. Physiol. 127:171–182). Here we show that, at rest, these regulation mechanisms are also involved in the modulation of calcium release activities. The enhancement of resting release activity may participate in the calcium overload observed in dystrophin-deficient myotubes, and our findings support the hypothesis of the regulatory role of mini-dystrophin on intracellular signaling.

Phagocytosis by Sertoli Cells: Analysis of Main Phagocytosis Steps by Confocal and Electron Microscopy

Sertoli cells were discovered in the seminiferous tubules by Enrico Sertoli in 1865 (Morgagni 7:31-33, 1865). Intense phagocytosis is, in the context of spermatogenesis cycle, morphologically the most noticeable function of Sertoli cells. In this chapter the major principles of phagocytosis machinery and its specificities in the seminiferous tubules will be briefly reviewed, guidelines of analysis of main phagocytosis steps by confocal and transmission electron microscopy will be described, and a simplified method to assess phagocytosis rate in routine experiments will be given.