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First published online 25 August 2004
doi: 10.1242/jcs.01335

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Dominant negative effect of connexin33 on gap junctional communication is mediated by connexin43 sequestration

¹ INSERM EMI 00-09, IFR 50, Faculté de Médecine, Avenue de Valombrose, 06107 Nice CEDEX 02, France
² LBSC, CNRS UMR 6558, Université de Poitiers, 86022 Poitiers CEDEX, France
³ INSERM U 364, IFR 50 Faculté de Médecine, 06107 Nice CEDEX 02, France
⁴ Université Paris V, 45 rue des Saint-Pères, 75006 Paris, France
⁵ GRIPL, EA 2675, Faculté de Médecine, 06107 Nice CEDEX 02, France
⁶ INSERM U 427, Faculté de Pharmacie, 75270 Paris, France

View larger version (52K): [in a new window]   Fig. 1. Presence of Cx33 in the rat testis. (A) Anti-Cx33 western blot analysis detected a band migrating at 33 kDa in lysates of testis but not of brain, heart, uterus and ovary. The anti-Cx43 antibody detected the presence of different phosphorylated Cx43 isoforms in all tissues (left panel). The numbers on the right indicate molecular mass (kDa). Immunolocalization of Cx33 in seminiferous tubules (right panel). Note that the Cx33 signal is only present in the basal compartment of seminiferous tubule (x120) whereas Cx43 is detected in both tubular and interstitial compartments (inset). (B,C) Characterization of the unphosphorylated status of Cx33. (B) Testis immunoprecipitates (Ip) with anti-Cx33 or anti-Cx43 antibodies were treated with (+) or without (-) alkaline phosphatase (AP) and analyzed by western blotting with Cx33 or Cx43 antibodies (left panels). Testis immunoprecipitates with anti-Cx33 were analyzed by western blot with Cx33, phosphotyrosine (Ptyr), phosphoserine (Pser) or phosphothreonine (Pthr) antibodies (right panel). (C) COS-7 cells were transfected with Cx43, Cx33-myc or empty vector (-) and labeled with [32P]orthophosphate. Cell lysates were then immunoprecipitated (Ip) with myc (left panel) or Cx43 (right panel) antibodies and separated by SDS-PAGE. The [32P] radiolabeled proteins were visualized by autoradiography and the presence of Cx33-myc and Cx43 was verified by western blotting with myc or Cx43 antibodies. The positions of Cx33 and Cx43 isoforms are shown by arrowheads. Figures are representative of three separate experiments. Open arrowheads indicate positions of unphosphorylated and phosphorylated forms of Cx43.

View larger version (60K): [in a new window]   Fig. 2. Selective association of Cx33 with Cx43. (A) Testis lysates were untreated (-) or immunoprecipitated (Ip) with anti-Cx33 or anti-Cx43 antibodies. Lysates and immunoprecipitates were analyzed by western blotting with Cx43, Cx33, Cx32 and Cx26 antibodies; all are detected in testis lysates at the predicted sizes of 39-45, 33, 32 and 26 kDa respectively. (B) Sertoli cells were stably transfected with vectors containing Cx33 and Cx33-myc cDNAs or empty vector as a control (-). Triton X-100 soluble fractions from Sertoli cells were immunoprecipitated with either Cx43 or myc antibodies and analyzed by western blotting with Cx33, Cx43 and myc antibodies. Open arrowheads indicate positions of unphosphorylated and phosphorylated forms of Cx43. Results are representative examples of three separate experiments.

View larger version (59K): [in a new window]   Fig. 3. Intracellular localization of Cx33/Cx43 complex in Sertoli cells. (A-F) Cells were transfected with the vector containing Cx33-myc cDNA (D,E,F) or vector alone (A,B,C). In the absence of Cx33-myc, Cx43 was localized as linear punctuate staining at appositional plasma membranes between adjacent Sertoli cells (A, arrowhead). In the presence of Cx33-myc, Cx43 was solely detected in the perinuclear region (D, arrowhead), the myc signal was localized in the same region (E, arrowhead). Merging Cx33 and Cx43 micrographs revealed colocalization of two signals in Cx33-myc transfected Sertoli cells (F). Images are representative of three separate experiments. Magnification, x600.

View larger version (95K): [in a new window]   Fig. 4. Intracellular localization of Cx33/Cx43 complex in COS-7 cells. (A-C) In the absence of Cx33 or Cx33-myc, Cx43 staining was punctuated and linear at appositional plasma membranes between adjacent COS-7 cells and myc was undetectable. (D-F) In cells transfected with Cx33-myc cDNA the signals for myc (D) and Cx33 (E) colocalized similarly around the nucleus (F). (G-I) Cells were transfected with Cx33 or (J-L) Cx33-myc cDNAs in combination with Cx43. In the presence of Cx33 or Cx33-myc, Cx43 was solely detected in the perinuclear region (G,J). Merging Cx33 (or myc) and Cx43 micrographs revealed colocalization of the two signals in Cx33 or Cx33-myc transfected cells (I,L). Images are representative of three separate experiments. Magnification, x800.

View larger version (108K): [in a new window]   Fig. 5. Sequestration of Cx43 within early endosomes in Cx33 transfected Sertoli cells. (A-T) Sertoli cells expressing Cx33 were transfected with EGFP-rab5, EGFP-rab7 or EGFP-lamp vectors or stained with antibodies specific of ER or Golgi apparatus. Fluorescent deconvolution microscopy analysis allowed the simultaneous detection of Cx33 (A,E,I,M,Q), Cx43 (B,F,J,N,R) with Endoplasmic Reticulum (C), Golgi apparatus (G), early endosomes (K), late endosomes (O) or lysosomes (S). (D,H,L,P,T) Merged images of Cx43, myc and EGFP localization. White spots indicate colocalization of the three signals. Note that the Cx33/Cx43 complex colocalizes mainly with EGFP-Rab5 (L) and weakly with ER (D). Results are representative of three separate experiments. Magnification, x800.

View larger version (63K): [in a new window]   Fig. 6. Inhibition of gap junctional coupling by Cx33. (A-F) Phase-contrast and immunofluorescence photomicrographs (x300) of Sertoli cells transfected with vector containing Cx33-myc cDNA (D,E,F) or vector alone (A,B,C) microinjected with a mixture of Lucifer yellow and rhodamine dextran. No transfer of dye was observed in Sertoli cells overexpressing Cx33 (D-F). By contrast, Lucifer yellow was transferred to adjacent cells in the control (C), whereas rhodamine dextran was localized only within the microinjected cell (B). Microinjected cells are identified with arrows. The average number (mean±s.d., n=20) of communicating cells in control (vector alone) and Cx33-myc transfected Sertoli cells is given in the table. *P<0.001.