Background Endogenous electric fields (EFs) play an essential role in guiding the coordinated collective migration of epidermal cells to the wound centre during wound healing. Although polarization of leadercells is essential for collective migration, the signal mechanisms responsible for the EF-induced polarization of leader cells under electrotactic collective migration remain unclear. This study aims to determine how the leader cells are polarized and coordinated during EF-guided collective migration of epidermal cell sheets. Methods Collective migration of the human epidermal monolayer (human immortalized keratinocytes HaCaT) under EFs was observed via time-lapse microscopy. The involvement of tetraspanin-29 (CD9) in EF-induced fibrous actin (F-actin) polarization of leader cells as well as electrotactic migration of the epidermal monolayer was evaluated by genetic manipulation. Blocking, rescue and co-culture experiments were conducted to explore the downstream signalling of CD9. Results EFs guided the coordinated collective migration of the epithelial monolayer to the anode, with dynamic formation of pseudopodia in leader cells at the front edge of the monolayer along the direction of migration. F-actin polarization, as expected, played an essential role in pseudopod formation in leader cells under EFs. By confocal microscopy, we found that CD9 was colocalized with F-actin on the cell surface and was particularly downregulated in leader cells by EFs. Interestingly, genetic overexpression of CD9 abolished EF-induced F-actin polarization in leader cells as well as collective migration in the epidermal monolayer. Mechanistically, CD9 determined the polarization of F-actin in leader cells by downregulating a disintegrin and metalloprotease 17/heparin-binding epidermal growth factor-like growth factor/epidermal growth factor receptor (ADAM17/HB-EGF/EGFR) signalling. The abolished polarization of leader cells due to CD9 overexpression could be restored in a co-culture monolayer where normal cells and CD9-overexpressing cells were mixed; however, this restoration was eliminated again by the addition of the HB-EGF-neutralizing antibody. Conclusion CD9 functions as a key regulator in the EF-guided collective migration of the epidermal monolayer by controlling and coordinating the polarization of leader cells through ADAM17/HB-EGF/EGFR signalling.

中文翻译：

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CD9 通过控制和协调前导细胞的极化来负调节表皮单层的集体趋电性

背景内源电场（EF）在伤口愈合过程中引导表皮细胞协调集体迁移到伤口中心方面发挥着重要作用。尽管前导细胞的极化对于集体迁移至关重要，但在电趋集体迁移下 EF 诱导前导细胞极化的信号机制仍不清楚。本研究旨在确定在 EF 引导的表皮细胞片集体迁移过程中，前导细胞如何极化和协调。方法通过延时显微镜观察 EF 下人表皮单层（人永生化角质形成细胞 HaCaT）的集体迁移。通过基因操作评估了 tetraspanin-29 (CD9) 在 EF 诱导的前导细胞纤维肌动蛋白 (F-actin) 极化以及表皮单层电趋化中的参与。进行阻断、救援和共培养实验来探索 CD9 的下游信号传导。结果 EF引导上皮单层向阳极协调集体迁移，单层前缘的前导细胞沿迁移方向动态形成伪足。正如预期的那样，F-肌动蛋白极化在 EF 下前导细胞的伪足形成中发挥了重要作用。通过共聚焦显微镜，我们发现 CD9 与 F-肌动蛋白共定位于细胞表面，并且在前导细胞中尤其受到 EF 的下调。有趣的是，CD9 的基因过度表达消除了 EF 诱导的前导细胞中 F-肌动蛋白极化以及表皮单层中的集体迁移。从机制上讲，CD9 通过下调解整合素和金属蛋白酶 17/肝素结合表皮生长因子样生长因子/表皮生长因子受体 (ADAM17/HB-EGF/EGFR) 信号传导来决定前导细胞中 F-肌动蛋白的极化。由于 CD9 过表达而消除的前导细胞极化可以在正常细胞和 CD9 过表达细胞混合的共培养单层中恢复；然而，通过添加 HB-EGF 中和抗体，这种恢复再次被消除。结论 CD9 通过 ADAM17/HB-EGF/EGFR 信号传导控制和协调前导细胞的极化，在 EF 引导的表皮单层集体迁移中发挥关键调节作用。