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First published online 13 September 2005
doi: 10.1242/jcs.02569

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Mechanisms of Cx43 and Cx26 transport to the plasma membrane and gap junction regeneration

Department of Anatomy and Cell Biology, University of Western Ontario, Dental Science Building, Rm. 00077, London, Ontario, Canada N6A 5C1

View larger version (41K): [in a new window]   Fig. 1. BICR-M1Rk cells assemble untagged and fluorescent-protein-tagged Cx26 and Cx43 into gap junctions via a trafficking pathway that is BFA-sensitive. Wild-type BICR-M1Rk (A,B) or BICR-M1Rk stably expressing Cx43-GFP (C,D), Cx26 (E,F) or Cx26-YFP (G,H) were immunolabeled for either Cx43 (A,B) or Cx26 (E,F); fluorescent-protein-tagged connexins were imaged without further processing. There was a prominent untagged or fluorescent-protein-tagged paranuclear-localized and plasma membrane-localized connexin population in untreated cells (A,C,E,G). However, following BFA treatment, all connexin variants were localized in an ER-like pattern (B,D,F,H) and gap junction plaques were not evident. Bar, 10 µm. (I) BFA-treatment of cells co-expressing Cx43 and Cx26 (BICR Cx26) dramatically inhibited dye coupling. Data expressed as mean number of cells receiving dye before or after BFA treatment±s.e.m.

View larger version (118K): [in a new window]   Fig. 2. Expression of mutant Sar1 disrupts ER-to-Golgi transport in BICR-M1Rk cells and inhibits Cx26 and Cx43 trafficking to the cell surface. BICR-M1Rk cells were transiently transfected with either Sar1-DsRed2 (A-C) or mutSar1-DsRed2 (D-F), and immunolabeled for the Golgi-resident protein GPP130 (A,D). The Golgi apparatus, as defined by GPP130 localization (arrows), remained intact in the presence of Sar1-DsRed2 but was disrupted in cells expressing mutSar1-DsRed2. BICR-M1Rk cells stably expressing Cx26-YFP were transiently transfected with Sar1-DsRed2 (G-J) or mutSar1-DsRed2 (K-N) and immunolabeled for Cx43. Anti-rabbit AMCA was used to visualize and separate the Cx43 signal from YFP and DsRed fluorescence. Sar1-DsRed2 did not disrupt the localization pattern of Cx43 or Cx26-YFP but mutSar1-DsRed2 caused their retention within the ER. Bars, 10 µm. WT, wild-type; MUT, mutant.

View larger version (61K): [in a new window]   Fig. 3. Cx26 and Cx43 exhibit similar kinetics and follow identical pathways during nascent gap junction biogenesis. BICR-M1Rk cells stably expressing Cx26 were treated with BFA for 6 hours and fixed at different times following BFA-washout prior to double immunofluorescently labeling for Cx26 (A,D,G,J,M; green) or Cx43 (B,E,H,K,N; red). In untreated conditions, Cx26 and Cx43 often co-localized to the same gap junctions (A-C, arrows) and within paranuclear regions of the cells consistent with localization to the Golgi apparatus (A-C, double arrows). Immediately after BFA removal, Cx26 and Cx43 exhibited an ER-like localization pattern (D-F; inset in D reveals a similar distribution of the resident Golgi protein, MG160). At 30 minutes post-BFA washout, Cx26 and Cx43 were localized in the reforming Golgi apparatus (G-I, double arrows; inset in G highlights paranuclear MG160), and by 90 minutes there was diffuse cell surface fluorescence exhibited by Cx26 and Cx43 (J-L, arrow; inset in J reveals paranuclear MG160 localization suggesting a well-organized Golgi apparatus). Also note the prominent population of Cx26 and Cx43 present within the Golgi apparatus (J-L, double arrows). By 2 hours (M-O), Cx26 and Cx43 localization was beginning to resemble control cells. Sparsely cultured cells were used in this experimental set to allow for the connexin distribution patterns to be assessed at sites of cell-cell contact and at free surfaces. Bar, 10 µm.

View larger version (37K): [in a new window]   Fig. 4. Nascent assembly of fluorescent-protein-tagged Cx26 and Cx43 follow the same kinetics and trafficking routes as untagged connexins. BICR-M1Rk cells stably expressing Cx26-YFP or Cx43-GFP were treated with BFA for 6 hours and fixed at different times after BFA removal. In untreated cells, Cx26-YFP and Cx43-GFP exhibited a paranuclear and cell surface localization pattern (A,B). Both Cx26-YFP and Cx43-GFP were localized within the ER-like compartment at 6 hours of BFA treatment and gap junction plaques were absent (C,D). At 30 minutes post-BFA treatment, both Cx26-YFP and Cx43-GFP were localized to the reformed Golgi apparatus (E,F) and by 60 minutes they were also detected at the cell surface as a diffuse rim of fluorescence (G,H). At 90 minutes, both connexins were visualized as cell surface gap junctions while prominent paranuclear populations of both connexins remained (I,J). Two hours after BFA washout, Cx26-YFP and Cx43-GFP localization resembled that of untreated cells (K,L). Bar, 10 µm.

View larger version (41K): [in a new window]   Fig. 5. Cx43-GFP and Cx26-YFP are transported from the Golgi apparatus in both vesicular- and tubular-like PGCs. Time-lapse images of BICR-M1Rk cells expressing Cx43-GFP (A1,A2) or Cx26-YFP (B1,B2) were obtained from 10 to 40 minutes after the removal of BFA. Movies 1 and 2 (see supplementary material) show that both Cx43-GFP and Cx26-YFP were predominantly in the ER initially (T=10) and were transported to paranuclear locations by 40 minutes (T=40), which reflects the re-organization of the Golgi apparatus. Cells imaged (T=84-90 minutes) after BFA removal revealed a paranuclear Golgi-like distribution of Cx43-GFP (C) and Cx26-YFP (D) with subpopulations of tagged connexins emanating from the Golgi in tubular-like structures (C,D, arrows) and vesicular structures (C,D, arrowheads). Tubular extensions and dynamic vesicle movement are best seen in Movies 3 and 4 (see supplementary material). (E) Cx26-YFP could be observed at the cell surface with a diffused rim appearance (double arrow) in cells imaged 90 minutes following BFA-washout. Movie 5 shows vesicles exiting distal elements of the Golgi apparatus and the appearance of bright clusters at cell surface that move rapidly in the plane of the membrane (see supplementary material). Bars, 10 µm.

View larger version (57K): [in a new window]   Fig. 6. Golgi-apparatus-localized Cx26 mutants actively exit the Golgi in the form of dynamic tubular extensions and vesicles. HBL-100 cells stably expressing D66K-GFP (A,C, green) or D66H-GFP (D,F, green) were labeled for the cis-Golgi resident protein, GPP130 (B,E,C,F; red). Both D66H-GFP and D66K-GFP exhibit a high degree of co-localization with the Golgi apparatus (C,F, note yellow color). Cells were counterstained with Hoechst 33342 to denote the nuclei (C,F, blue). HBL-100 cells expressing D66H-GFP were time-lapse imaged following photobleaching of the Golgi population (series G, box indicates photobleached region). A new population of D66H-GFP migrated to the photobleached Golgi apparatus with a 40% recovery of fluorescence in 1 hour (based on the pre-bleached area being 100%). Tubular extensions were observed exiting the Golgi apparatus of HBL-100 cells expressing D66H-GFP (series H, arrows). In series I, the area surrounding the Golgi pool of D66K-GFP mutant was photobleached and time-lapse imaged. Highly dynamic vesicles were observed to emanate from the Golgi apparatus within 15 minutes (series I, arrows and supplementary material Movie 6). Bars, 10 µm.

View larger version (35K): [in a new window]   Fig. 7. Differential mobility of fluorescent-protein-tagged Cx26 and Cx43 within assembled gap junctions. Central regions of gap junction plaques in BICR-M1Rk cells, composed of Cx26-YFP (A-D) or Cx43-GFP (E-H), were photobleached and the fluorescent recovery in the photobleached area was monitored. Note the rapid recovery of Cx26-YFP within 200-400 seconds but little recovery was observed in Cx43-GFP photobleached regions of gap junction plaques. Further FRAP analysis revealed that gap junctions composed of Cx26-YFP had a substantially larger mobile fraction population than Cx43-GFP gap junctions (I). Bar, 5 µm.

View larger version (98K): [in a new window]   Fig. 8. Steady-state regeneration of Cx26-GFP gap junctions is sensitive to BFA but not nocodazole. NRK cells stably expressing Cx26-GFP were time-lapse imaged following photobleaching of a series of gap junctions (A,D,G, box indicates photobleached area). Cx26-GFP fluorescence steadily recovered within the photobleached area (G, box). ER-to-Golgi transport was disrupted by a 45 minute pre-incubation with BFA followed by FRAP in the presence of BFA (B,E,H, box indicates photobleached area). After 1 hour, there was no detectable Cx26-GFP within the photobleached region of interest (H, box). To disrupt microtubules, nocodazole was added to the cell culture 45 minutes prior to photobleaching of gap junctions in the continued presence of nocodazole (C,F,I). There was significant recovery of Cx26-GFP to the region of interest after 1 hour (I, box), comparable with that occurring under control conditions. Panel inserts represent higher magnifications of the photobleached regions of interest. Experimental sets were repeated seven times with similar results. Bar, 10 µm.

View larger version (84K): [in a new window]   Fig. 9. Steady-state regeneration of Cx43-GFP gap junctions is sensitive to BFA and nocodazole. NRK cells stably expressing Cx43-GFP were time-lapse imaged following photobleaching of a series of gap junctions in the presence of BFA or nocodazole. Control cells revealed substantial recruitment of Cx43-GFP to the photobleached gap junctions after 1 hour (A,D,G) but this level of recovery was not observed in cells treated with BFA (B,E,H) or nocodazole (C,F,I). Panel inserts represent higher magnifications of the photobleached regions of interest. Experimental sets were repeated six times with similar results. Bar, 10 µm.

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