The epidermal growth factor receptor (EGFR) governs pivotal signaling pathways in cell proliferation and survival, with mutations implicated in numerous cancers. The organization of EGFR on the plasma membrane (PM) is influenced by the lipids and the cortical actin (CA) cytoskeleton. Despite the presence of a putative actin-binding domain (ABD) spanning 13 residues, a direct interaction between EGFR and CA has not been definitively established. While disrupting the cytoskeleton can impact EGFR behavior, suggesting a connection, the influence of the static actin cytoskeleton has been found to be indirect. Here, we investigate the potential interaction between EGFR and CA, as well as the extent to which CA regulates EGFR’s distribution on the PM using SRRF’n’TIRF, a spatiotemporal super-resolution microscopy technique that provides sub-100 nm resolution and ms-scale dynamics from the same data set. To label CA, we constructed PMT-mEGFP-F-tractin, which combines an inner leaflet targeting domain PMT, fluorescent probe mEGFP, and the actin-binding protein F-tractin. In addition to EGFR-mEGFP, we included two control constructs: 1) an ABD deletion mutant, EGFRΔABD-mEGFP serving as a negative control and 2) EGFR-mApple-F-tractin, where F-tractin is fused to the C-terminus of EGFR-mApple, serving as the positive control. We find that EGFR-mEGFP and EGFRΔABD-mEGFP show similar membrane dynamics, implying that EGFR-mEGFP dynamics and organization are independent of CA. EGFR dynamics show CA dependence when F-tractin is anchored to the cytoplasmic tail. Together, our results demonstrate that EGFR does not directly interact with the CA in its resting and activated state.

中文翻译：

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EGFR 不直接与皮质肌动蛋白相互作用：SRRF'n'TIRF 研究


表皮生长因子受体 （EGFR） 控制细胞增殖和存活的关键信号通路，突变与许多癌症有关。质膜 （PM） 上 EGFR 的组织受脂质和皮质肌动蛋白 （CA） 细胞骨架的影响。尽管存在跨越 13 个残基的推定肌动蛋白结合结构域 （ABD），但 EGFR 和 CA 之间的直接相互作用尚未明确确定。虽然破坏细胞骨架会影响 EGFR 行为，表明存在联系，但已发现静态肌动蛋白细胞骨架的影响是间接的。在这里，我们使用 SRRF'n'TIRF 研究了 EGFR 和 CA 之间的潜在相互作用，以及 CA 在多大程度上调节 EGFR 在 PM 上的分布，SRRF'n'TIRF 是一种时空超分辨率显微镜技术，可提供来自同一数据集的亚 100 nm 分辨率和 ms 级动力学。为了标记 CA，我们构建了 PMT-mEGFP-F-tractin，它结合了内小叶靶向结构域 PMT、荧光探针 mEGFP 和肌动蛋白结合蛋白 F-tractin。除了 EGFR-mEGFP，我们还包括两个对照构建体：1） ABD 缺失突变体，EGFRΔABD-mEGFP 作为阴性对照和 2） EGFR-mApple-F-tractin，其中 F-tractin 与 EGFR-mApple 的 C 端融合，作为阳性对照。我们发现 EGFR-mEGFP 和 EGFRΔABD-mEGFP 表现出相似的膜动力学，这意味着 EGFR-mEGFP 动力学和组织与 CA 无关。当 F 牵引蛋白锚定到细胞质尾部时，EGFR 动力学显示 CA 依赖性。总之，我们的结果表明 EGFR 不直接与处于静息和激活状态的 CA 相互作用。