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First published online 22 November 2005
doi: 10.1242/jcs.02700

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Hyper-adhesion in desmosomes: its regulation in wound healing and possible relationship to cadherin crystal structure

David R. Garrod, Mohamed Y. Berika^*, William F. Bardsley, David Holmes and Lydia Tabernero

Faculty of Life Sciences, Michael Smith Building, Oxford Road, University of Manchester, Manchester, M13 9PT, UK

View larger version (170K): [in a new window]   Fig. 1. Transmission electron micrographs of desmosomes in mouse epidermis. Normal (A) and wound-edge (B) mouse epidermis after exposure to calcium-free Dulbecco's minimum essential medium with 10% chelated foetal bovine serum and 3 mM EGTA (LCM-EGTA) for 6 hours (A) and 1 hour (B). Note that the desmosomes in A are intact and two that are sectioned precisely transversely exhibit midline structure, whereas in B the desmosomal halves (blue arrows) have lost adhesion and separated. Thus the desmosomes in A are calcium independent and those in B are calcium dependent. Quantification is shown in Table 1. (C-F) Comparison between desmosomes in unwounded and wound-edge epidermis. (C) Desmosome in unwounded epidermis showing characteristic midline structure. (D-F) Examples of transversely sectioned desmosomes from wound-edge epidermis showing absence of midlines. Quantification is shown in Table 2. Bars, 0.1 µm.

View larger version (93K): [in a new window]   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).

View larger version (139K): [in a new window]   Fig. 3. Electron micrographs of intracytoplasmic desmosomes from mouse skin wounds. Images within 10 cell diameters (A-D) or 40-50 cell diameters from the wound edge (E,F), showing intracytoplasmic desmosomes (blue arrows). B is an enlargement of the area enclosed within the square in A. Note the absence of desmosomes from the cell surface membranes in all images. Black arrows indicate possible membrane fragments associated with some desmosomes. Bars, 100 nm.

View larger version (44K): [in a new window]   Fig. 4. Schematic representation of the Dsc2 ectodomain model 3D array, generated from the crystallographic structure of C-cadherin (Boggon et al., 2002). In the centre the desmosomal interspace is represented as a cylinder, showing the mid-line formed by trans and cis interactions between molecules on opposed cell surfaces. Monomers from one cell surface are coloured in red and monomers from the opposed cell surface are coloured in blue. The midline is aligned with the x-axis of the cylinder (coincident with the x-axis in the crystallographic lattice). Rotation of the 3D array around the x-axis by 90° (top) shows a regular lattice with distances between rows of molecules of 73.85 Å and distances of 75.14 Å between layers. Rotations around the z-axis produce four different views, all of them with a midline dense zone (bottom). These are: strand dimer 1 at z=0°; `boat' at z=30°, strand dimer 2 (inverse form of strand dimer 1) at z=60° and `zipper' at z=120°. The cis and trans interfaces between distal domains are indicated by arrows.

View larger version (121K): [in a new window]   Fig. 5. (A) Transmission electron micrographs of a desmosome from guinea-pig heart after infiltration with lanthanum chloride cut in transverse section. Note how the mid line has a zigzag appearance. This arises because the dense particles (P) in one row are staggered with respect to those in the opposite row, and the pale central lamella (L) has side arms that extend between the particles. (B) En face view of lanthanum-infiltrated desmosome from guinea-pig heart showing a series of alternating dark and light parallel lines of 75 Å periodicity quadratic array of lanthanum-filled spaces. D, desmosome; J, gap junction. Images in A and B are reproduced from Rayns et al. (1969) by copyright permission of the Rockefeller University Press. (C) Extracted central region from image B after high-pass filtering and masking. (D) Autocorrelation image of C to show period structure. This shows both a strong lattice repeat of 75 Å and a second weaker lattice repeat of 72 Å with a direction about 85° to the first. (E) Power spectrum of image C with intensity peaks from the lattice structure circled. (F) Fourier-filtered image using a lattice mask based on the first order peaks shown in E. Bars, 0.1 µm (A,B); 200 Å (C,D,F); reciprocal space scale bar in E, 1/100 Å–1.

View larger version (38K): [in a new window]   Fig. 6. (A) Detailed representation of the cis and trans interfaces in the 3D array packing generated with the Dsc2 model using the C-cadherin ectodomain structure (Boggon et al., 2002). Monomers from opposed cell surfaces (yellow and blue) form trans-interactions mediated by Trp2 in the N-terminal domain (EC1' and EC1'' respectively). The Trp side chain is shown binding into the hydrophobic pocket of the opposed N-terminal domain. Cis interactions occur between the EC1' domain (yellow) and the linker region between the EC2 and EC3 domains on another monomer from the same cell surface (red). (B) Zoom view of the linker region. The EC1' domain (yellow) from one monomer inserts the ß-helix region, as a wedge, into the cavity formed between the EC2 and EC3 domains of a neighbouring molecule (red). This cis interface blocks access to the most-exposed calcium-binding site occupied by Ca3. The other two calcium ions, Ca1 and Ca2 are bound deeper into the core of domain EC2. Ca1 and Ca2 are coordinated by five ligand groups from different side chains and two groups from the main chain (not shown). Ca3 is coordinated only by four ligand groups from side chains in EC2 and EC3. The figure was prepared using SETOR.

View larger version (28K): [in a new window]   Fig. 7. Summary of events believed to lead to the downregulation of desmosomal adhesion in wounded epidermis. (A) In normal epidermis PKC is diffusely distributed in keratinocyte cytoplasm, and desmosomes have an organised structure and are Ca2+ independent. (B) Following wounding PKC associates with the desmosomal plaque. (C,D) PKC mediates phosphorylation of desmosomal plaque component(s). (E) A transmembrane signal generates a less-organised arrangement of the desmosomal cadherins and onset of Ca2+ dependence. (F) Whole desmosomes are internalised by wound-edge cells probably initially in association with membrane vesicles (red arrow).

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