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实时成像方法与荧光标记的蛋白质来分析桥粒组分的功能，这是至关重要的表皮角质形成细胞之间的细胞粘附。使用该技术，我们评估了空间（细胞定位）以及时间（每小时的变化）运动的桥粒成分在培养的角质形成细胞中的天疱疮中的抗桥粒芯糖蛋白3（Dsg 3）IgG抗体。用腺病毒介导的方法将绿色荧光蛋白标记的角蛋白14（EGFP-K14）转化正常人表皮角质形成细胞（NHEK），在细胞质中形成网状荧光角蛋白纤维。当我们将培养基中的Ca浓度转移到更高的浓度时，荧光角蛋白形成向细胞-细胞接触位点的插入。将抗Dsg 3 IgG抗体与EGFP-K14表达的NHEK细胞孵育导致角质形成细胞的脱离以及Dsg 3的内化，伴随着荧光角蛋白纤维从细胞-细胞接触部位的缩回。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，Dsg 3以及EGFP-K14响应于抗Dsg 3 IgG的动力学受到抗Dsg 3 IgG对培养的角质形成细胞的细胞-细胞粘附破坏的致病强度的积极影响。这些发现一起表明，我们的实时成像方法将提供新的见解，不仅阐明天疱疮水疱形成的分子机制，而且表皮角质形成细胞的细胞-细胞粘附功能。

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.