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Multiple pathways in the trafficking and assembly of connexin 26, 32 and 43 into gap junction intercellular communication channels

Patricia E. M. Martin^*,, Geraldine Blundell, Shoeb Ahmad, Rachel J. Errington and W. Howard Evans

Department of Medical Biochemistry, University of Wales College of Medicine, Heath Park, Cardiff CF14 4XN, UK
^* Present address: Department of Radiology, Wales Heart Research Institute, University of Wales College of Medicine, Heath Park, Cardiff CF14 4XN, UK

View larger version (73K): [in a new window]   Fig. 1. Assignment of colocalisation coefficient to Cx-GFP expressing cells. Real time trafficking of Cx26-GFP was recorded every 30 seconds over a 30 minute time period. Time points 1 (30 seconds, t1), 30 (15 minutes, t2) and 60 (30 minutes, t3) were assigned red (A), green (B) and blue (C) channels, respectively. Merging the three images gives a view of the vesicular trafficking in the cell (D). (E) Representative fluorogram of the extent of colocalisation of red and blue channels where the line of identity is indicated (see Materials and Methods for details). Ce, colocalisation coefficient. Bar, 5 µm. Arrows indicate vesicular movement between the three time points.

View larger version (27K): [in a new window]   Fig. 2. The effect of inhibitors of the secretory pathway and cytoskeleton on intercellular transfer of Lucifer yellow. HeLa cells expressing the various connexin proteins were assessed for their ability to transfer Lucifer yellow across gap junctions as described in Materials and Methods. The results are expressed as the percentage of cells transferring dye to >2 neighbouring cells ±s.e.m. At least 90 cells were injected per experiment, n=3. The effect of 15°C and cytochalasin D on cells expressing wtCx32 and Cx32I28L was not determined (Nd). Statistical significance was determined by Student t-test analysis. P<0.05 was considered significant. *P<0.05; **P<0.001; *** P<0.0005.

View larger version (124K): [in a new window]   Fig. 3. The effect of Brefeldin A, Nocodazole and Cytochalasin D on Golgi, microtubules and actin filaments. Confluent monolayers of HeLa cells were fixed and stained with a primary antibody to ERGIC (p58) under control conditions (A) and following 5 hours treatment with 5 µg/ml BFA (B); microtubule integrity was monitored by use of antibodies to ß-tubulin under normal conditions (C) and following 5 hours treatment with 20 µg/ml nocodazole (D). The structural integrity of actin filaments was determined by staining with pholloidin before (E) and after 5 hours treatment with 1 µg/ml cytochalasin D (F). Bar, 10 µm.

View larger version (63K): [in a new window]   Fig. 4. The effect of BFA on Cx32-CFP and Cx26-YFP in co-transfected COS-7 cells. COS-7 cells were co-transfected with cDNA encoding Cx32-CFP and Cx26-YFP. Cells expressing both proteins were examined before and after BFA treatment. (A-C) No drug treatment. Cx32-CFP (A), Cx26-YFP (B) and merged image (C) showing colocalisation of both proteins especially at a presumed gap junction. (D-F) Cells treated with BFA for 6 hours. Cx32-CFP (D), Cx26-YFP (E) and merged image (F) showing absence of Cx32-CFP at the plasma membrane. For the purpose of image analysis CFP was assigned to the red channel and YFP to the green channel, resulting colocalisation in the merged image is yellow. Bar, 10 µm. Arrows indicate targeting to gap junctions.

View larger version (111K): [in a new window]   Fig. 5. The effect of BFA on the trafficking of mutant Cx32I28L. COS-7 cells were transfected with cDNA encoding Cx32I28L and 48 hours post-transfection cells were fixed and stained with an antibody against the carboxyl tail of Cx32 (Gap34R). (A) Cx32I28L under normal conditions. Similar images were obtained for wtCx32 and mutant Cx32V206I. (B) Mutant Cx32I28L 6 hours post-treatment with BFA. Arrow shows retention of gap junctions at the plasma membrane. (C) Cx32V206I 6 hours post treatment with BFA. Note that Cx32 staining was intracellular. (D) wtCx26 6 hours following nocodazole treatment showing that Cx26 was intracellular. (E) wt Cx32 6 hours following nocodazole treatment. Arrow shows gap junctions. (F) Mutant Cx32I28L 6 hours following nocdoazole treatment showing limited plasma membrane but much increased intracellular staining. D and F show a reduction in plasma membrane staining. Bar, 10 µm, Arrows indicate targeting to the gap junction. Parallel staining of the cells with p58 or ß-tubulin was used to confirm that disassembly of the Golgi following BFA treatment and shattering of the microtubules following nocodazole treatment had occurred in a similar manner to that shown in Fig. 3 (not shown).

View larger version (110K): [in a new window]   Fig. 6. The effect of BFA and nocodazole on vesicular trafficking of Cx43-GFP and Cx26-GFP. Images were analysed as described in Fig. 1 before and after treatment of the cells with BFA or nocodazole for 5 hours. Red, green and blue vesicles indicate vesicular trafficking, a white image indicates colocalisation of the three time points. Cx43-GFP (A) and Cx26-GFP (B) under normal conditions; Cx43-GFP (C) and Cx26-GFP (D) following BFA treatment; Cx43-GFP (E) and Cx26-GFP (F) following nocodazole treatment. Arrows point to gap junctions at the plasma membrane.

View larger version (22K): [in a new window]   Fig. 7. Co- and post-transitional insertion of wild-type and mutated connexins into microsomal membranes. WtCx32 and Cx26, Cx32I28L or Cx32V206I were synthesised in the absence of microsomal membranes (-), or with microsomes added co-translationally (co) or post-translationally (po). For co- and post-translational studies, membranes were separated from free connexins by centrifugation through a sucrose cushion, washed in an alkaline buffer and then analysed for the presence of membrane-inserted connexins by SDS-PAGE. Lanes 1-3, Cx26; lanes 4-6, Cx32; lanes 7-9, Cx32I28L; lanes 10-12, Cx32V206I. The faster migrating bands (approx. 5 kDa less than full length connexins) are caused by abnormal proteolytic cleavage occurring in vitro due to a signal peptidase (Falk et al., 1997).