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Fig. 2. PKC is colocalised with desmoplakin in wound-edge epithelium after wounding. (A) In unwounded epidermis PKC (green) is diffusely distributed in the cytoplasm of mouse keratinocytes and is not colocalised with desmoplakin (red). (B) By contrast, in wound-edge epithelium 72 hours post wounding, PKC and desmoplakin show substantial colocalisation (yellow). The white arrow indicates the wound edge. Quantification of the spread of PKC-desmoplakin colocalisation from the wound edge with time is shown in Table 3A. (C-F) Localisation of PKC to desmosomal plaques by immuno-gold labelling of ultra-thin cryosections. Unwounded epidermis (C,E) and wound-edge epidermis (D, F) 48 hours after wounding. The desmosomes (red arrowheads in C and D) are unlabelled in normal epidermis, but the desmosomes and surrounding cytoplasm are heavily labelled in wound epidermis. Label in D that is not clearly associated with the two transversely sectioned desmosomes may be associated with desmosomal plaques cut en face or with intermediate filaments. Note none of the desmosomes show midlines because these structures are not visible by this technique (see North et al., 1999). Gold particles are 10 nm in diameter. Quantification of immuno-gold labelling in normal and 72 hour wound-edge epidermis is shown in Table 3B. (G,H) Distribution of PKC in wound desmosomes. A low-resolution map of the desmosomal plaque from quantitative analysis of the distributions of gold particles after immuno-labelling with specific antibodies is published (North et al., 1999). To determine the distribution of PKC in the desmosomal plaque, the distribution of PKC labelling was similarly analysed in desmosomes that were double-labelled for desmoplakin C-terminus as an internal control. Particle distances from the cell membrane were determined. The peak of desmoplakin labelling was at 48.9 nm from the membrane (H), in good agreement with previous results (North et al., 1999). Deconvolution of the particle distribution suggested the presence of two PKC peaks, one at 0.32-1.08 nm from the cell membrane and a much more diffuse peak at 18.2-21.8 nm from the membrane (G). The latter peak suggests localisation within the outer dense plaque of the desmosome (North et al., 1999). Bars, 5 µm (A,B); 0.1 µm (C,D); 30 nm (E,F).

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