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First published online 25 November 2008
doi: 10.1242/jcs.033373

Journal of Cell Science 121, 4069-4078 (2008)
Published by The Company of Biologists 2008
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Molecular reorganization of Cx43, Zo-1 and Src complexes during the endocytosis of gap junction plaques in response to a non-genomic carcinogen

Jérome Gilleron1,2, Céline Fiorini1,2, Diane Carette1,2, Christiane Avondet1,2,*, Matthias M. Falk3, Dominique Segretain1,2 and Georges Pointis1,2,{ddagger}

1 INSERM U 895, Team 5 Physiopathologic control of germ cell proliferation: genomic and non genomic mechanisms, Université Paris Descartes, 45 rue des Saint-Pères, 75006, Paris, France
2 Centre de Mediterranéen de Médecine Moléculaire (C3M), 151 route Saint-Antoine de Ginestière BP 2 3194, 06204 Nice cedex 3, France
3 Department of Biological Sciences, Lehigh University, 111 Research Drive, Iacocca Hall, Bethlehem, PA 18015, USA


Figure 1
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  		Fig. 1. Kinetics of endocytosis of gap junction plaques. (A) Analysis of Cx43-GFP (green) in 42GPA9 Sertoli cells 24 hours after transfection. The two upper panels show Golgi localization of (left) Cx43-GFP (anti-CTR433 antibody) and (right) its presence at the plasma membrane level, identified through the occludin immunosignal. The two lower panels show the presence of Cx43-GFP in (left) early endosomes (cells transfected with the expression plasmid for Rab5a-wt-mRFPC1) and (right) in lysosomes (anti-Lamp2 antibody). As shown by arrows and in the insets, the cytoplasmic Cx43-GFP spots (green) strongly colocalized (yellow) with the markers of the Golgi complex, early endosomes and lysosomes. Cell nuclei are identified by DAPI staining. (B) Analysis of Cx43-GFP (green) gap junction plaque in 42GPA9 Sertoli cells incubated for 2 hours in the presence or absence of 50 µM HCH (known to stimulate Cx43 internalization). Notice that, in HCH-treated cells, all green fluorescence is detected within endocytic vesicles, whereas in control cells gap junction plaques are present between communicating cells (arrows). Cell nuclei (blue) are also shown. Upper panels are phase-contrast micrographs, dashed lines indicate the position of the cell membranes. (C) Time-lapse video microscopy of internalization of Cx43-GFP gap junction plaques in control and HCH-treated cells. The dynamic phases of annular gap junction formation are illustrated. t0, gap junction plaque between two adjacent cells; t15 min, beginning of the internalization process of the plaque; t30 min, annular gap junction formation. Dotted lines indicate plasma membranes. (D) Sertoli cells transfected with Cx43-GFP were incubated for different time periods in the presence or absence of HCH. The number of cells with Cx43-GFP gap junction plaques was quantified under each condition and expressed in percent vs total transfected cells identified by the presence of GFP at the plasma membrane and into the cytoplasm. *P<0.05 and **P<0.01, significantly different from untreated cells.

 

Figure 2
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  		Fig. 2. Involvement of activated Src in Cx43-GFP gap junction plaque endocytic internalization. (A-D) Time-dependent effects of HCH on activation of Src and the MAPK pathway (i.e. ERK, p38 and JNK). Sertoli cells were cultured in the presence of 50 µM HCH for increasing times (0, 3.5, 7.5, 30 and 60 minutes). At the indicated times, cells were lysed and levels of activated (i.e. phosphorylated) Src (A), ERK (C), p38 and JNK (D) were assessed by western blotting as described in Materials and Methods (top panels of A, C and D). Total Src and total ERK1/2 levels were also identified (A, C lower panels). Densitometric scanning of Src phosphorylation during exposure to HCH is indicated in B. Similar quantification was carried out for phosphorylation of p42 and p44 (ERK2 and ERK1, respectively) (data not shown). A representative blot of three different experiments is shown for each experiment. * P<0.05 significantly different from time 0.

 

Figure 3
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  		Fig. 3. Role of activated Src by HCH in the number of gap junction plaques and functionality. 42GPA9 Sertoli cells transfected with Cx43-GFP were incubated in the presence or absence of HCH for different time periods (0-30 minutes), with or without PP2 or PD98059. (A) The number of cells with Cx43-GFP gap junction plaques was quantified under each condition and expressed in percent vs total transfected cells identified by the presence of GFP at the plasma membrane and within the cytoplasm. (B) Dye coupling analysis, as described in Materials and Methods, in control and HCH-treated 42GPA9 Sertoli cells. Notice that calcein fluorescence has noticeably recovered in control cells but not in HCH-exposed cells. (C) Dye-transfer efficiency is expressed as percentage of fluorescence recovery after calcein photobleaching in Sertoli cells cultured for the same time periods (0-30 minutes) in the presence of HCH with or without PP2 or PD98059. *P<0.05 and **P<0.01, significantly different from untreated cells.

 

Figure 4
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  		Fig. 4. Subcellular localization of Cx43 and Src in response to HCH exposure. Sertoli cells were treated with 50 µM HCH for 10 minutes in the presence or absence of 30 µM PP2 or 30 µM PP3. Cx43 and Src were then detected by immunofluorescence. Notice that HCH treatment resulted in both aberrant cytoplasmic localization of Cx43 and recruitment of Src to the plasma membrane (arrows), is totally prevented by the presence of PP2 but not of PP3. In both situations, proteins relocation are easily noticeable by membranous lines (arrows) or cytoplasmic dots (arrowheads). Images are a typical example of three separate experiments.

 

Figure 5
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  		Fig. 5. Changes in Cx43–ZO-1 and Cx43-Src associations after exposure to HCH. (A) Lysates from 42GPA9 Sertoli cells that had been incubated with or without 50 µM HCH were immunoprecipitated (Ip) with antibodies against ZO-1, Src or Cx43. Immunoprecipitates were then analyzed by western blotting using antibodies against ZO-1, Src and Cx43. Bands of ZO-1, Src and Cx43 were detected at the predicted molecular masses of 120, 55 and 43 kDa, respectively. In the presence of HCH, levels of ZO-1 associated with Cx43 decreased when lysates were immunoprecipited with either ZO-1 or Cx43. After Cx43 Ip, low but detectable levels of Src were associated with Cx43 in HCH-treated cells, but were totally absent in control cells. (B) Lysates from 42GPA9 Sertoli cells subjected for 1 hour to 50 µM HCH with or without 30 µM PP2 were immunoprecipitated with antibodies against Cx43 or ZO-1. Cx43 and ZO-1 immunoprecipitates were then analyzed by western blotting for P-Src (p-Src) using anti-Src [pY418] antibody. For each experiment in A and B, a representative blot of three different experiments is shown. Relative optical densities of the bands in arbitrary units are presented in the right panels. Results are the average of three different experiments. *P<0.05, **P<0.01 significantly different from untreated cells.

 

Figure 6
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  		Fig. 6. Close molecular interactions between Cx43, ZO-1 and Src during HCH-induced endocytic internalization of Cx43-GFP gap junction plaques. (A) Percentage of two adjacent cells in which one Cx43-GFP transfected (T) cell is in contact with a wild-type cell expressing endogenous Cx43 (Wt), or in which the two cells express Cx43-GFP (T/T). The transfected cells were identified by the presence of GFP within cells. (B,C) Localization of immunoreactive ZO-1 between two adjacent cells that (B) express Cx43-GFP, or between (C) one cell that expresses Cx43-GFP (T) and a wild-type cell (Wt) that expresses only endogenous Cx43. Dashed lines correspond to the zone of contact between the two adjacent cells. (D-K) High-resolution deconvolution fluorescence microscopy analysis of the interaction of Cx43-GFP with ZO-1 and Src during the internalization of gap junction plaques in response to 50 µM HCH. (D) Side view of a gap junction plaque (green) decorated on both sides by ZO-1 (red). Colocalization is shown in yellow. (E) Presence of ZO-1 only on one side of the plaque after HCH treatment, indicating the beginning of the plaque endocytosis. (F) Localization of ZO-1 on the internal side of the gap junction plaque during internalization. (G) View of an early Cx43-GFP annular gap junction associated with ZO-1 in its center. (H) Cx43-GFP gap junction plaque (green) and distribution of Src (red) in control cells. (I) Side view of a gap junction plaque associated at one side with Src after HCH exposure. Colocalization is shown in yellow. (J) At the beginning of plaque internalization the presence of Src (yellow) was mainly detected on the external side of the gap junction plaque. (K) View of an early Cx43-GFP annular gap junction associated in its periphery with Src (yellow). Images are representative of five different experiments.

 

Figure 7
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  		Fig. 7. Immunolocalization of ZO-1 and Src in annular gap junctions and Amira volume-reconstructions. (A) Upper panels: localization of ZO-1 (red) in serial sections (0, 1, 2, 3 µm) of a Cx43-GFP annular gap junction (AGJ) characterized by the presence of green fluorescence. Notice that colocalization (shown in yellow) occurs inside the annular gap junction. Lower panels: localization of Src (red) in serial sections of a Cx43-GFP-containing AGJ (green). Notice that colocalization (shown in yellow) occurs outside the AGJ. (B,C) Amira volume-reconstructions of the interactions between ZO-1 and Cx43, and Src and Cx43 in AGJ. (B) Tilting angles (–45°, 0°, +45°) of an entire AGJ reconstructed with Amira software from Cx43-GFP (green) and ZO-1 (red) experiments demonstrate the absence of ZO-1 outside of the structure (upper panels). When `cutting open' part of the AGJ, ZO-1 clearly appeared accumulated inside (lower panels). Semi-quantitative analysis confirmed the internal localization of ZO-1 (right panel). (C) Amira volume-reconstructions of interaction between Src and Cx43 in annular gap junctions. Tilting angles (–45°, 0°, +45°) of a complete AGJ reconstruct with Amira software from Cx43-GFP (green) and Src (red) experiments demonstrating strict outside localization of the kinase on the AGJ (arrows and insets). Even if some Src signals are present just near the AGJ, i.e. without making contact, others are tightly associated with the AGJ (arrows). Cross sections of rotated open AGJ show no Src signal inside the AGJ. Semi-quantitative analysis confirmed Src labeling at the outside (right panel) versus ZO-1 labeling at the inside. Values are the means ± s.e.m. **P<0.01, n=100.

 

Figure 8
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  		Fig. 8. Illustration of the molecular dynamics of the interactions of Cx43, ZO-1 and Src during endocytic internalization of gap junction plaques in response to treatment with HCH. If a sufficient amount of activated Src is present in pathological tissues or in cells that were treated with carcinogen, dissociation of the Cx43–ZO-1 complex occurs after the kinase Src associates with Cx43, rapidly resulting in both internalization of the gap junction plaque and formation of the annular gap junction. The reduced level of ZO-1 on one side of the Cx43 gap junction plaque at the beginning of internalization could be indicative of the cell that endocytes the gap junction plaque.

 

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© The Company of Biologists Ltd 2008