When considering connexin expression and regulation, the epidermis of the skin is one of the most complex tissues found in mammals even though it largely contains a single cell type, the keratinocyte. In the rodent epidermis, up to 9 connexin family members have been detected at the mRNA level. Many of these connexins are temporally and spatially regulated in coordination with keratinocyte progenitor cell differentiation and migration from the stratum basale to form the stratum spinosum and stratum granulosum layers before finally forming the stratum corneum. Cx43 is the principal connexin found in basal keratinocytes and to a lesser degree found in keratinocytes that have begun to differentiate where Cx26, Cx30 and Cx31 become prevalent. Here we show that the CRISPR-Cas9 ablation of Cx43 reduces overall gap junction coupling in monolayer cultures of rat epidermal keratinocytes (REKs) and dysregulates the differentiation of REKs when grown in organotypic cultures. Natively found in differentiated keratinocytes, Cx31 readily assembles into gap junctions when expressed in REKs where it can extensively co-assemble into the same gap junctions with co-expressed Cx30. Time-lapse imaging indicated that many Cx31 gap junctions are mobile within the plasma membrane undergoing both fusion and fission events. Finally, the persistence of pre-existing Cx31 gap junctions in the presence of the protein trafficking blocker, brefeldin A, is longer than that found for Cx43 gap junctions indicating that it has a distinctly different life expectancy in REKs. Collectively, this study highlights the importance of Cx43 in rodent keratinocyte differentiation and suggests that Cx31 acquires life-cycle properties that are distinct from Cx43.

中文翻译：

---

Cx31 和 Cx43 在具有分化能力的啮齿动物角质形成细胞中的比较分析

在考虑连接蛋白的表达和调节时，皮肤的表皮是哺乳动物中发现的最复杂的组织之一，尽管它主要包含单一细胞类型，即角质形成细胞。在啮齿动物表皮中，在 mRNA 水平上检测到多达 9 个连接蛋白家族成员。在最终形成角质层之前，许多这些连接蛋白与角质形成细胞祖细胞的分化和从基底层迁移以形成棘层和颗粒层层相协调，在时间和空间上受到调节。Cx43 是在基底角质形成细胞中发现的主要连接蛋白，在开始分化的角质形成细胞中发现的程度较低，其中 Cx26、Cx30 和 Cx31 变得普遍。在这里，我们展示了 Cx43 的 CRISPR-Cas9 消融降低了大鼠表皮角质形成细胞 (REK) 单层培养物中的整体间隙连接耦合，并在器官型培养物中生长时失调了 REK 的分化。Cx31 天然存在于分化的角质形成细胞中，当在 REK 中表达时，它很容易组装成间隙连接，在那里它可以与共同表达的 Cx30 广泛地共同组装成相同的间隙连接。延时成像表明许多 Cx31 间隙连接在经历融合和裂变事件的质膜内是可移动的。最后，在存在蛋白质运输阻滞剂 brefeldin A 的情况下，预先存在的 Cx31 间隙连接的持久性比 Cx43 间隙连接的持久性更长，表明它在 REK 中具有明显不同的预期寿命。总的来说，