Department of Biomedicine, University of Basel, Basel, Switzerland.
Faculty of Medicine, Ludwig-Maximilians-Universität Munich, Munich, Germany.
Acta Physiol (Oxf). 2021 Apr;231(4):e13609. doi: 10.1111/apha.13609. Epub 2021 Jan 19.
Desmoplakin (Dp) is a crucial component of the desmosome, a supramolecular cell junction complex anchoring intermediate filaments. The mechanisms how Dp modulates cell-cell adhesion are only partially understood. Here, we studied the impact of Dp on the function of desmosomal adhesion molecules, desmosome turnover and intercellular adhesion.
CRISPR/Cas9 was used for gene editing of human keratinocytes which were characterized by Western blot and immunostaining. Desmosomal ultrastructure and function were assessed by electron microscopy and cell adhesion assays. Single molecule binding properties and localization of desmosomal cadherins were studied by atomic force microscopy and super-resolution imaging.
Knockout (ko) of Dp impaired cell cohesion to drastically higher extents as ko of another desmosomal protein, plakoglobin (Pg). In contrast to Pg ko, desmosomes were completely absent in Dp ko. Binding properties of the desmosomal adhesion molecules desmocollin (Dsc) 3 and desmoglein (Dsg) 3 remained unaltered under loss of Dp. Dp was required for assembling desmosomal cadherins into large clusters, as Dsg2 and Dsc3, adhesion molecules primarily localized within desmosomes, were redistributed into small puncta in the cell membrane of Dp ko cells. Additional silencing of desmosomal cadherins in Dp ko did not further increase loss of intercellular adhesion.
Our data demonstrate that Dp is essential for desmosome formation but does not influence intercellular adhesion on the level of individual cadherin binding properties. Rather, macro-clustering of desmosomal adhesion molecules through Dp is crucial. These results may help to better understand severe diseases which are caused by Dp dysfunction.
桥粒斑蛋白（Dp）是桥粒这一细胞间连接复合物的重要组成部分，该复合物锚定中间丝。Dp 调节细胞间黏附的机制尚未完全阐明。本研究旨在探讨 Dp 对桥粒黏附分子功能、桥粒周转和细胞间黏附的影响。
利用 CRISPR/Cas9 基因编辑技术对人角质形成细胞进行基因编辑，通过 Western blot 和免疫染色进行鉴定。利用电子显微镜和细胞黏附实验评估桥粒的超微结构和功能。利用原子力显微镜和超分辨率成像研究桥粒钙黏蛋白的单分子结合特性和定位。
与桥粒斑蛋白（Pg）缺失相比，Dp 缺失显著损害细胞间黏附，导致细胞聚集成更大的团块。与 Pg 缺失不同的是，Dp 缺失的细胞完全没有桥粒。在 Dp 缺失的情况下，桥粒黏附分子桥粒胶蛋白（Dsg）3 和桥粒糖蛋白（Dsg）3 的结合特性保持不变。Dp 对于桥粒钙黏蛋白组装成大簇是必需的，因为 Dsg2 和 Dsc3 等主要定位于桥粒的黏附分子在 Dp 缺失细胞的质膜中重新分布成小的点状结构。在 Dp 缺失的情况下进一步沉默桥粒钙黏蛋白并没有进一步增加细胞间黏附的丧失。
我们的数据表明，Dp 对于桥粒的形成是必需的，但不会影响单个钙黏蛋白结合特性的细胞间黏附。相反，Dp 通过桥粒黏附分子的巨聚类对于桥粒的形成是至关重要的。这些结果可能有助于更好地理解由 Dp 功能障碍引起的严重疾病。
