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First published online 15 March 2005
doi: 10.1242/jcs.01733

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Connexin interaction patterns in keratinocytes revealed morphologically and by FRET analysis

¹ Centre for Cutaneous Research, Institute of Cell and Molecular Science, Barts and the London School of Medicine and Dentistry, Queen Mary, University of London, 4 Newark Street, London E1 2AT, UK
² Cancer Research UK London Research Institute, Lincoln's Inn Fields Laboratories, 44 Lincoln's Inn Fields, London WC2A 3PX, UK

View larger version (44K): [in a new window]   Fig. 1. A u-adFRET assay. Keratinocytes were co-transfected with (wt)Cx30-ECFP (cyan pseudocolour, donor) and (wt)Cx26-EYFP (yellow pseudocolour, acceptor). (A) Expression patterns of (wt)Cx30-ECFP (left), (wt)Cx26-EYFP (middle) and the merged image (right) before bleaching (top row) and the changes of fluorescence intensity in areas of interest (c to h) after bleaching (bottom row) show enhanced fluorescence intensity of donor (cyan pseudocolour, left) and reduced fluorescence intensity of acceptor (yellow pseudocolour, middle). Cyan and yellow pseudocolour look-up tables were designed to produce white in the merged image when perfectly colocalised. Areas labelled a and b were used as a background reference and an unbleached reference respectively. (B) Example profiles (from region g) of relative expression concentrations of donor and acceptor (top) and the changes of fluorescence intensity in donor and acceptor before and after bleaching (bottom). Bar, 10 µm.

View larger version (112K): [in a new window]   Fig. 2. Immunofluorescence staining of connexins in skin. Green fluorescence represents anti-connexin antibody (A to F) and red fluorescence (C and E) represents anti-ß-actin antibody. (A) Cx30.3 and (B) Cx31 both localised in the stratum granulosum of control epidermis. (C) Cx26 expression in the hair follicle (hf) and sweat gland (sg) of control skin (note that Cx26 is undetectable in normal interfollicular epidermis). (D) Cx30 expression was clearly localised at the plasma membrane of keratinocytes in the upper epidermal layers of control epidermis. (E) Cx26 expression in the sweat glands (sg) of skin from the KID patient heterozygous for the D50N mutation in Cx26 was comparable to that seen in control skin. (F) Interfollicular skin obtained from the HED patient heterozygous for the G11R mutation in Cx30 was stained by Cx30 antibody and revealed a sparse staining pattern with some protein detectable at the plasma membrane. An enlargement of the small, boxed area is shown. Asterisk indicates probable localisation of aggregated Cx30 protein at the plasma membrane in keratinocytes of the stratum granulosum. de, position of dermal-epidermal junction. Bar, 20 µm.

View larger version (77K): [in a new window]   Fig. 3. FRET images of homomeric connexin formation in NEB1 keratinocytes. The first three rows show pairs of cells co-transfected with (wt)Cx26-ECFP and (wt)Cx26-EYFP, (wt)Cx30-ECFP and (wt)Cx30-EYFP, and (wt)Cx31-ECFP and (wt)Cx31-EYFP. These co-transfections clearly show large gap junction-like aggregates of the fusion proteins at the cell-cell interface. In contrast the three cells in contact presented in the fourth row co-transfected with (wt)Cx30.3-ECFP and (wt)Cx30.3-EYFP did not show any aggregates at the plasma membrane. Left-hand panels are (wt)Cx-ECFP images, middle panels, (wt)Cx-EYFP images and right-hand panels, merged images. Bar, 10 µm.

View larger version (98K): [in a new window]   Fig. 4. Images of heteromeric connexin formations in NEB1 keratinocytes. Cells were co-transfected with different pairs of wild-type connexins (Cx) fused with ECFP or EYFP. Left-hand panels, Cx-ECFP images, middle panels, Cx-EYFP images and right-hand panels, merged images. Co-transfection with different combinations of (wt)Cx26, (wt)Cx30 and (wt)Cx31 clearly show large gap junction-like aggregates of the fusion proteins at the cell-cell interface. In contrast, none of the combinations with (wt)Cx30.3 showed any aggregates at the plasma membrane. Bar, 10 µm.

View larger version (124K): [in a new window]   Fig. 5. Images of localisation patterns of mutant (D50N)Cx26 and wild-type connexins in co-transfected NEB1 keratinocytes. Cells were co-transfected with a combination of two constructs at a ratio of 1:1. Unlike (D50N)Cx26 on its own, any combination of (D50N)Cx26 with wild-type connexin produced a punctate pattern of localisation in the cytoplasm. Left-hand panels are Cx-ECFP images, middle panels Cx-EYFP images and right-hand panels merged images of Cx-ECFP and Cx-EYFP. Bar, 10 µm.

View larger version (117K): [in a new window]   Fig. 6. Images of localisation patterns of mutant (G11R)Cx30 and wild-type connexins in co-transfected NEB1 keratinocytes. Cells were co-transfected with a combination of two constructs at a ratio of 1:1. Unlike (G11R)Cx30 on its own, any combination of (G11R)Cx30 with wild-type connexin produced a punctate pattern of localisation in the cytoplasm. Additionally combinations of (G11R)Cx30 with (wt)Cx26 or (wt)Cx30 produced small aggregates at cell-cell contacts. Left-hand panels are Cx-ECFP images, middle panels, Cx-EYFP images and right-hand panels, merged images of Cx-ECFP and Cx-EYFP. Bar, 10 µm.

View larger version (27K): [in a new window]   Fig. 7. Mean FRET efficiency as a function of relative concentration ratio of donor/acceptor (Cx-ECFP/Cx-EYFP) and acceptor fluorescence intensity level for different combinations of connexins. The decrease of FRET efficiency with the increase of donor/acceptor concentration ratio, but its insensitivity to the increase of absolute acceptor level, indicate that donors and acceptors are clustered into connexons.

View larger version (47K): [in a new window]   Fig. 8. Immunoprecipitation analysis. Cells were transfected with a Cx-EGFP construct (first two lanes) or pairs of Cx-EGFP constructs immunoprecipitated with either Cx26 or Cx30.3 antibodies. Western blotting was performed using an anti-EGFP-HRP antibody to detect any Cx-EGFP chimeric protein present. The Cx26 antibody immunoprecipitated Cx26-EGFP+Cx30-EGFP or Cx26-EGFP+Cx30.3-EGFP. In addition, the Cx30.3 antibody immunoprecipitated Cx26-EGFP+Cx30.3-EGFP. The size of the Cx26-EGFP band is 53 kDa and both Cx30-EGFP and Cx30.3-EGFP were 56 kDa. The interaction between Cx26 and Cx30 appears to be much stronger although this may be due to the higher levels of Cx26 and Cx30 expression compared to Cx30.3 seen in these experiments.

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