Department of Dermatology and Department of Cell Biology, Duke University, Durham, NC 27710.
Mol Biol Cell. 2020 May 15;31(11):1140-1153. doi: 10.1091/mbc.E19-09-0542. Epub 2020 Apr 2.
Desmosomes are cell-cell adhesions necessary for the maintenance of tissue integrity in the skin and heart. While the core components of desmosomes have been identified, peripheral components that modulate canonical or noncanonical desmosome functions still remain largely unexplored. Here we used targeted proximity labeling approaches to further elaborate the desmosome proteome in epidermal keratinocytes. Quantitative mass spectrometry analysis identified all core desmosomal proteins while uncovering a diverse array of new constituents with broad molecular functions. By individually targeting the inner and outer dense plaques, we defined proteins enriched within these subcompartments. We validated a number of these novel desmosome-associated proteins and find that many are membrane proximal proteins that show a dependence on functional desmosomes for their cortical localization. We further explored the mechanism of localization and function of two novel desmosome-associated adaptor proteins enriched in the desmosome proteome, Crk and Crk-like (CrkL). These proteins interacted with Dsg1 and rely on Dsg1 and desmoplakin for robust cortical localization. Epidermal deletion of both Crk and CrkL resulted in perinatal lethality with defects in desmosome morphology and keratin organization, thus demonstrating the utility of this dataset in identifying novel proteins required for desmosome-dependent epidermal integrity.
桥粒是细胞间黏附所必需的，对于维持皮肤和心脏组织的完整性至关重要。尽管桥粒的核心成分已经确定，但调节经典或非经典桥粒功能的外围成分在很大程度上仍未被探索。在这里，我们使用靶向邻近标记方法进一步阐述了表皮角质形成细胞中的桥粒蛋白组。定量质谱分析鉴定了所有核心桥粒蛋白，同时揭示了广泛具有广泛分子功能的各种新成分。通过分别靶向内致密斑和外致密斑，我们确定了这些亚区中富含的蛋白质。我们验证了其中许多新型桥粒相关蛋白，并发现许多是膜近端蛋白，它们的皮质定位依赖于功能性桥粒。我们进一步探讨了两个在桥粒蛋白组中富集的新型桥粒相关衔接蛋白（Crk 和 Crk-like，即 CrkL）的定位和功能的机制。这些蛋白与 Dsg1 相互作用，并且依赖于 Dsg1 和桥粒斑蛋白来实现其皮质定位。表皮中 Crk 和 CrkL 的缺失导致围产期致死，桥粒形态和角蛋白组织缺陷，因此证明了该数据集在识别桥粒依赖性表皮完整性所需的新型蛋白中的应用价值。
