Gorzkowska Jagoda, Kozak Wiktoria, Bobis-Wozowicz Sylwia, Cherepashuk Ivan, Madeja Zbigniew, Lasota Sławomir
Faculty of Biochemistry, Biophysics and Biotechnology, Department of Cell Biology, Jagiellonian University, Gronostajowa 7, Kraków, 30-387, Poland.
Cell Commun Signal. 2025 Apr 8;23(1):173. doi: 10.1186/s12964-025-02165-4.
Electrotaxis, the directed cell movement in direct current electric field (dcEF), is crucial for wound healing and development. We recently proposed a biphasic electrotaxis mechanism, where an initial rapid response is driven by ionic mechanisms, while redistribution of membrane components come into play during prolonged exposure to dcEF.
To verify this hypothesis, we studied the redistribution dynamics of EGFR, PDGFRα/β, and TGFβR1 in dcEF. For this purpose, we utilized cells transfected with plasmids encoding fluorescently tagged receptors, which were exposed to dcEF in a custom-designed electrotactic chamber. Fluorescent images were captured using wide-field or TIRF microscopy, enabling precise quantitative analysis of receptor redistribution. Additionally, the functional significance of these selected receptors in electrotaxis was evaluated by silencing their expression using an siRNA library.
Although EGFR moved immediately to cathode after dcEF application, maximum distribution asymmetry was reached after 30-40 min. This process was more efficient at higher dcEF intensities, specifically, asymmetry was greater at 3 V/cm compared to 1 V/cm, consistent with the biphasic mechanism observed only under the stronger dcEF. Additionally, redistribution was more effective under alkaline conditions and near the cell base, but decreased when glass was coated with poly-L-lysine, indicating electroosmosis as a key factor. Importantly, EGFR redistribution did not correlate with the rapid reaction of 3T3 cells to dcEF reversal, which occurred within 1-2 min, when receptor orientation was not yet reversed. PDGFRα exhibited similar but less marked cathodal redistribution, while PDGFRβ and TGFβR1 did not redistribute. siRNA knockdown experiments confirmed the importance of EGFR and ErbB4 in the electrotaxis. EGFR's role was largely ligand-independent, and it had a significant impact on the response of 3T3 cells to dcEF during the first hour of the experiment, but was not involved in the fastest response, which was Kir-dependent.
Our study suggests that EGFR redistribution may play a role in the early stages and partially contribute to the long-term electrotaxis of 3T3 fibroblasts. However, this mechanism alone does not fully explain rapid responses to dcEF orientation changes indicating a more complex, multimodal mechanism of electrotaxis in these cells.
电趋性，即细胞在直流电场（dcEF）中的定向移动，对伤口愈合和发育至关重要。我们最近提出了一种双相电趋性机制，其中初始的快速反应由离子机制驱动，而膜成分的重新分布在长时间暴露于dcEF期间发挥作用。
为了验证这一假设，我们研究了dcEF中表皮生长因子受体（EGFR）、血小板衍生生长因子受体α/β（PDGFRα/β）和转化生长因子β受体1（TGFβR1）的重新分布动态。为此，我们利用转染了编码荧光标记受体质粒的细胞，将其置于定制设计的电趋性室中暴露于dcEF。使用宽场或全内反射荧光显微镜（TIRF）拍摄荧光图像，从而能够对受体重新分布进行精确的定量分析。此外，通过使用小干扰RNA（siRNA）文库沉默这些选定受体的表达，评估了它们在电趋性中的功能意义。
虽然在施加dcEF后EGFR立即向阴极移动，但在30 - 40分钟后达到最大分布不对称。在较高的dcEF强度下，这个过程更有效，具体而言，与1V/cm相比，在3V/cm时不对称性更大，这与仅在较强dcEF下观察到的双相机制一致。此外，在碱性条件下和细胞基部附近重新分布更有效，但当玻璃用聚-L-赖氨酸包被时重新分布减少，表明电渗是一个关键因素。重要的是，EGFR的重新分布与3T3细胞对dcEF反转的快速反应不相关，后者发生在1 - 2分钟内，此时受体方向尚未反转。PDGFRα表现出类似但不太明显的阴极重新分布，而PDGFRβ和TGFβR1没有重新分布。siRNA敲低实验证实了EGFR和表皮生长因子受体4（ErbB4）在电趋性中的重要性。EGFR的作用在很大程度上不依赖配体，并且在实验的第一个小时内对3T3细胞对dcEF的反应有显著影响，但不参与最快的反应，后者依赖于内向整流钾通道（Kir）。
我们的研究表明，EGFR重新分布可能在早期阶段发挥作用，并部分促成3T3成纤维细胞的长期电趋性。然而，仅这一机制并不能完全解释对dcEF方向变化的快速反应，这表明这些细胞中电趋性存在更复杂的多模式机制。
