Real-time imaging analysis of cell adhesion molecules of epidermal keratinocytes

点击翻译按钮获取中文摘要

In this study, we developed a novel real-time imaging method with fluorescent-tagged proteins to analyze the functions of desmosomal components, which is critical for cell-cell adhesion between epidermal keratinocytes. Using the technique, we assessed spatial (cellular localization) as well as temporal (variation per hour) movements of the desmosomal components in cultured keratinocytes in response to anti-desmoglein 3 (Dsg3) IgG antibodies in pemphigus. Transformation of GFP-tagged keratin 14 (EGFP-K14) to Normal Human Epidermal Keratinocyte (NHEK) using adenovirus expression system resulted in a reticular network formation of fluorescent keratin fibers in the cytoplasm of NHEK cells. When we shifted the Ca concentration in the culture media to a higher concentration, the fluorescent keratins formed insertion towards cell-cell contact sites. Incubation of anti-Dsg3 IgG antibodies to EGFP-K14-expressed NHEK cells resulted in detachment of keratinocytes as well as in internalization of Dsg3 accompanied by retraction of fluorescent keratin fibers from cell-cell contact sites. These phenomena were determined by either time-lapse imaging analysis or real-time imaging analysis. Furthermore, dynamics of Dsg3 as well as EGFP-K14 in response to anti-Dsg3 IgG were positively influenced by pathogenic strength of the anti-Dsg3 IgG on disruption of cell-cell adhesion of cultured keratinocytes. These findings taken together suggested that our real-time imaging method will give novel insights not only to elucidate the molecular mechanisms for blister formation in pemphigus, but also to cell-cell adhesive functions of epidermal keratinocytes.