, Projet Yggdrasil de collaboration franco
, SFBBM bourse pour le 35 ème congrès FEBS
, FEBS bourse pour le workshop "spatiotemporal dynamics of cell signalling
,
, Initiateur et co-manageur du "réseau français sur l'AMPc" (avec Dr. P. Vincent)
, Elu par l'European Placenta Group Business Meeting comme faisant parti du "EPG Planning Committee
,
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Defect of villous cytotrophoblast differentiation into syncytiotrophoblast in Down syndrome, J Clin Endocrinol Metab, vol.85, pp.3700-3707, 2000. ,
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, Biolog) and eluted with cAMP. To remove any bound or unbound cAMP, the purified recombinant proteins were dialysed extensively against a buffer containing 20 mM MOPS, pH 7.0 and 150 mM NaCl. Protein concentrations were determined using the Bradford protein assay and SDS-PAGE (10% gels) using BSA as a standard. His-ezrin, GST-ezrin were expressed and purified as previously described, Bovine RI? (RI? and RI?-flag tagged) and human RII?, 2011.
Prior to pull down, 50 µl dry volume of immobilized cAMP beads (? 300 nmol of cAMP) were washed with 1 ml of PBS buffer. For control, beads blocked by ethanolamine were used in a parallel identical pulldown procedure. Prior to the pull-down assays, cell lysates were incubated with 10 mM ADP/GDP for 15 min at 4°C to reduce nonspecific binding, mainly contributed by ADP-and GDP-binding protein, Pull-down assays The cAMP-coupled-agarose beads, 2006. ,
USA) connected to a linear quadrupole ion trap -Orbitrap (LTQ Orbitrap) mass spectrometer (ThermoElectron, Bremen, Germany) equipped with a nanoelectrospray ion source. For liquid chromatography separation we used an Acclaim, Orbitrap mass spectrometry Dried peptides were dissolved in 10 µl 1% formic acid in water and 5 µl were injected onto an LC/MS system consisting of a Dionex Ultimate 3000 nano-LC system ,
, Survey full scan MS spectra (from m/z 300 to 2,000) were acquired in the Orbitrap with resolution R = 60,000 at m/z 400 (after accumulation to a target of 1,000,000 charges in the LTQ). The method used allowed sequential isolation of the most intense ions, up to six, depending on signal intensity, for fragmentation on the linear ion trap using collisionally induced dissociation at a target value of 100,000 charges, The mass spectrometer was operated in the data-dependent mode to automatically switch between Orbitrap-MS and LTQ-MS/MS acquisition, 2010.
, Gap-Fluorecence Recovery After Photobleaching (gap-FRAP) experiments
Cells were plated on eight-well chamber slides (Ibidi) at density of 100,000 cells per well and treated with 8-beta-glycyrrhetinic acid (?-GA) loaded with cell-permeable anchoring disruptor peptides or PKI peptide or transfected ezrin siRNA alone or concomitantly with mammalian expression vectors encoding GFP-ezrin # , GFP-ezrin * , GFP-ezrin * -AKBmut or GFP-ezrin * -D510I-R517V or transfected Cx43 siRNA alone or with mammalian expression vectors encoding GFP-Cx43 ? , GFP-Cx43 ? R370E or either with combinations of substittuions in the Cx43 PKA phosphorylation sites as described above and listed in Fig S4F. Cells were next loaded with calcein red-orange AM (Invitrogen) and images were acquired with a camera (EM-CCD eVolve, pixel: (16 µm) 2 ). In each experiment, one labeled, isolated cell was left unbleached as a refence for the loss of fluorescence due to repeated scanning and dye leakage. The microscope was controlled by expression upon ezrin silencing, PLA with ezrin and PKA (RI? and RII?) during trophoblast fusion and a summary of GFP-Cx43 variants. Fig. S5 shows the identification and characterization of minimal residues binding motifs in ezrin and Cx43. Video 1 corresponds to Fig 5B and shows the effect of gap junction inhibitor (beta-GA), Arg-tagged protein kinase inhibitor (PKI) or Arg-tagged anchoring disruptors RIAD or SuperAKAP-IS and their scrambled controls on gap junction communication in live cells examined by calcein transfer as fluorescence recovery after photobleaching (gap-FRAP). Video 2 corresponds to Fig. 7B and shows trophoblasts transfected with scrambled ezrin siRNA or ezrin siRNA with or without co-transfection with GFP-ezrin # , GFP-ezrin*, GFP-ezrin*-AKBmut plasmid or GFP-ezrin*-D510I-R517V on gap junction communication in live cells examined by calcein transfer as gap-FRAP. Video 3 corresponds to Fig. 9B and shows trophoblasts transfected with scrambled siRNA or Cx43 siRNA with or without co, Gap junction communication was quantitatively followed in live cells by gap-FRAP experiments as previously described, 1986. ,
, LIST OF ABBREVIATIONS Abbreviations used in this paper: 6-Bnz-cAMP, N6-benzoyladenosine 3',5'-cyclic monophosphate; 8-Br-cAMP, 8-bromoadenosine 3',5'-cyclic monophosphate
,
, A kinase anchoring protein, AKAP
,
, GA, 18 beta-glycyrrhetinic acid, vol.7
exchange protein activated by cAMP; ERM, ezrin-radixin-moesin; gap-FRAP, gap junction communication by fluorescence recovery after photobleaching; GCM1, glial cell missing 1 protein; hCG, human chorionic gonadotropin ,
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Red underscore: ezrin and Cx43 interacting regions. (C) Filters with peptides encompassing the minimal binding motifs of ezrin and Cx43 with or without substitutions (blue underscore) were overlayed with purified GST-Cx43 or GST-ezrin ,
, Trophoblasts transfected with ezrin specific-siRNA or scrambled control and co-transfected with siRNA insensitive GFP-ezrin D510I-R517V (GFP-ezrin* D510I-R517V) and cultured for 72 h
, Histograms represent remaining mononuclear cells and fusion index (left panels) and level of hCG and hPL (right panels) secreted to the medium. Scale bar: 30 µm. Results are expressed as the mean ± SEM of n = 3 independent experiments (ns for non significant, vol.001, p.0
, Ezrin bound to Cx43 is necessary for PKA control of gap junction communication. (A) Lysates from trophoblasts transfected with ezrin siRNA and GFP-ezrin*, GFP-ezrin*-AKBmut or GFP-ezrin*-D510I-R517V plasmids were subjected to immunoprecipitation with GFP antibody, vol.7
Dotted lines indicate lanes combined from single gel and exposure. (B) Trophoblasts were transfected with scrambled ezrin siRNA or ezrin siRNA with or without co-transfection with GFPezrin # , GFP-ezrin*, GFP-ezrin*-AKBmut plasmid or GFP-ezrin*-D510I-R517V, stimulated with hCG and subjected to gap-FRAP analysis. Left: calcein fluorescence intensity (F) transfer in individual trophoblasts (asterisk) mapped to pseudocolors as indicated by the color-scale bar, Scale bar: 10 µm. Right: calcein percent fluorescence ratio Ft/Fi ,
, Cell fusion is controlled by PKA-mediated phosphorylation of Cx43 through ezrin anchoring. (A) Trophoblasts were transfected with Cx43 siRNA or scrambled control alone or together with mammalian expression vectors directing expression of siRNA-resistant GFP-Cx43 (GFP-Cx43 ? ), GFP-Cx43 R370E (GFP-Cx43 ? -R370E), or GFP-Cx43 with substititions in the Cx43-PKA phosphosite (GFP-Cx43 ? -6SD, GFP-Cx43 ? -6SD-R370E, GFP-Cx43 ? -6SA, GFP-Cx43 ? -6SA, vol.8
, Cells with Cx43 knock down and/or reconstitution as in A were immunostained for desmoplakin (red) and nuclei were counterstained with DAPI. Scale bar: 30 µm. (C) Histograms represent percentage of mononuclear cells and fusion indices at 72 h of culture as in A and B. (D) The GFP-Cx43 ? -R370E fusion protein with out ability to bind ezrin and with individual phospho-or dephospho-mimicking S to D and S to A substitutions in residues 364, 365, 368 or all 3 (3SD and 3SAindicated, stimulated with hCG and subjected to GFPimmunoprecipitation (A) and gap-FRAP analysis (B). (C) Calcein fluorescence intensity (F) transfer in individual trophoblasts (asterisk) mapped to pseudocolors as indicated by the color-scale bar, R370E), cultured for another 72 h and subjected to immunoblot analysis with indicated antibodies. Note: higher molecular weight of GFP-Cx43 than ezrin is due to the GFP-tag. (B)
, Upon hCG stimulation
, Cx43 (E'), ezrin (F'), RI? (G'), RII? (H'), ZO-1 (I'), SP1 (J'), Cx43-SP1 (K') and ezrin-SP1 (L') and nuclei were counterstained with DAPI (D-K'). (M-T) Cells were separately immunostained for mIgG, rIgG, D-L) and cells were stained with pairs of antibodies or individual antibody alone as depicted in the figure: mIgG-rIgG (D')
, Scale bar: 15 µm. Histograms show the intensity of the red dot signals normalized by the number of nuclei (right panels; mean ± SEM of n = 3 independent experiments. (B, C, E) Trophoblast were treated with 8-Br-cAMP or hCG alone or together with anchor disrupting peptides or their scrambled controls (B), Supplemental Figure S4: Effect of anchoring disruptor peptides, ezrin silencing and Cx43 variants on cell adhesion
, Scale bars: 15 µm. Results are expressed as the mean ± SEM of n = 6 (B, C) or n=3 (E) independent experiments (*** p < 0.001). (D) Immunoblot analysis of desmoplakin, E-cadherin and actin levels in trophoblasts transfected with ezrin specific-siRNA or scrambled control and co-transfected with siRNA insensitive GFP-ezrin (GFP-ezrin*), siRNA insensitive GFP-ezrin AKB mutated (GFP-ezrin*-AKBmut) or siRNA insensitive GFP-ezrin* with mutation in the ezrin binding domain (GFP-ezrin*-D510I-R517V) after 72 h of culture. (F) Table describing the GFP-Cx43 variants, Cells were next stained with a pair of antibodies to Cx43 or GFP and desmoplakin (DSK) and subjected to proximity ligation in vitro assay (PLA)
, Supplemental Figure S5: Identification and characterization of minimal residues binding motifs in ezrin and Cx43. (A) The full length human ezrin (upper panels) and Cx43