Revisiting Type VII Collagen Mislocalisation in Dystrophic Epidermolysis Bullosa Using High-Resolution Imaging

Type VII collagen (COL7), a major component of anchoring fibrils, is essential for dermal-epidermal adhesion and pathogenic variants in COL7A1, encoding COL7, cause dystrophic epidermolysis bullosa (DEB). In normal skin, COL7 is localised just beneath the lamina densa. In contrast, previous immunoelectron microscopy (IEM) studies of DEB skin with residual COL7 expression demonstrated aberrant localisation of COL7 above the lamina densa and hemidesmosomes of epidermal basal keratinocytes, possibly reflecting impaired secretion of defective COL7. However, conventional microscopy techniques lack sufficient spatial resolution to clearly visualise individual protein distributions and resolve this finding. Here, we revisit the abnormal localisation of COL7 using two higher-resolution approaches: structured illumination microscopy (SIM) and expansion microscopy (ExM). In normal human skin, the COL7 NC1 domain colocalised with type IV collagen (COL4), a surrogate marker of the lamina densa and was consistently detected beneath integrin α6 (ITGA6), a hemidesmosomal marker. In DEB skin with residual COL7 expression, COL7 was detected above ITGA6 within basal keratinocytes, recapitulating previous IEM observations. SIM quantitatively confirmed significant BMZ disorganisation in DEB, whereas ExM showed a consistent but non-significant trend, likely due to variability in expansion factors. These findings demonstrate that high-resolution fluorescence imaging can reproduce classic IEM observations of COL7 mislocalisation, suggesting its potential as a more accessible complementary approach for morphologic assessment of DEB.