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

在这项研究中，我们开发了一种新的荧光标记蛋白实时成像方法来分析桥粒组分的功能，桥粒组分对表皮角质形成细胞之间的细胞粘附至关重要。使用该技术，我们评估了在天疱疮中，培养的角化细胞中桥粒体成分对抗桥粒蛋白3 (Dsg3) IgG抗体的响应的空间（细胞定位）和时间（每小时变化）运动。利用腺病毒表达系统将gfp标记的角蛋白14 （EGFP-K14）转化为正常人表皮角质细胞（NHEK），在NHEK细胞的细胞质中形成网状的荧光角蛋白纤维。当我们将培养基中的Ca浓度提高到较高浓度时，荧光角蛋白向细胞-细胞接触部位形成插入。将抗Dsg3 IgG抗体与egfp - k14表达的NHEK细胞孵育，导致角质形成细胞脱离，Dsg3内化，并伴有荧光角蛋白纤维从细胞-细胞接触部位缩回。这些现象是通过延时成像分析或实时成像分析确定的。此外，Dsg3和EGFP-K14对抗Dsg3 IgG的响应动态受到抗Dsg3 IgG的致病强度对培养的角质形成细胞细胞粘附破坏的积极影响。综上所述，我们的实时成像方法不仅将为阐明天疱疮水疱形成的分子机制提供新的见解，而且还将为表皮角质形成细胞的细胞粘附功能提供新的见解。

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.