Centre for Micro-Photonics, Faculty of Science, Engineering and Technology, Swinburne University of Technology, Hawthorn, VIC, Australia.
Adv Exp Med Biol. 2018;1112:41-52. doi: 10.1007/978-981-13-3065-0_4.
The epidermal growth factor receptor (EGFR) is an important cell surface receptor in normal physiology and disease. Recent work has shown that EGF-gold nanoparticle conjugates can influence cell behaviour, but the underlying mechanism at the receptor quaternary structural level remains poorly understood.In the present work, the cluster density and cluster size of activated (phosphorylated) EGFR clusters in HeLa cells were determined with photobleaching image correlation spectroscopy. EGFR activation was probed via immunofluorescence-detected phosphorylation of tyrosines (pY-mAb) located in the kinase domain of EGFR (Y845) and at the EGFR cytoplasmic tail (Y1173). Cell activation was probed via nuclear extracellular-regulated kinase (ERK) phosphorylation. The cluster size of activated EGFR was 1.3-2.4 pY-mAb/cluster in unstimulated HeLa cells. EGF or nanorod treatment led to an increase in EGFR oligomers containing multiple phosphotyrosines (>2 phosphotyrosines per EGFR oligomer, average cluster size range = 3-5 pY-mAb/cluster) which paralleled increases in nuclear p-ERK. In contrast, EGF-nanorods decreased the contribution from higher-order phospho-clusters and decreased nuclear p-ERK relative to the nanorod control. These studies provide direct evidence that targeted nanotechnology can manipulate receptor organization and lead to changes in receptor activation and subsequent signalling processes.
表皮生长因子受体 (EGFR) 是正常生理和疾病中的重要细胞表面受体。最近的研究表明，EGF-金纳米粒子缀合物可以影响细胞行为，但受体四级结构水平的潜在机制仍知之甚少。在本工作中，通过光漂白图像相关光谱法确定了 HeLa 细胞中激活（磷酸化）EGFR 簇的团簇密度和团簇大小。通过免疫荧光检测 EGFR 激酶结构域 (Y845) 和 EGFR 胞质尾部 (Y1173) 处的磷酸化酪氨酸 (pY-mAb) 探测 EGFR 的激活。通过核细胞外调节激酶 (ERK) 磷酸化探测细胞激活。未刺激的 HeLa 细胞中，激活的 EGFR 的簇大小为 1.3-2.4 pY-mAb/簇。EGF 或纳米棒处理导致含有多个磷酸酪氨酸的 EGFR 寡聚体增加（每个 EGFR 寡聚体多于 2 个磷酸酪氨酸，平均簇大小范围=3-5 pY-mAb/簇），与核 p-ERK 的增加平行。相比之下，EGF-纳米棒与纳米棒对照相比，降低了来自更高阶磷酸簇的贡献，并降低了核 p-ERK。这些研究提供了直接证据，表明靶向纳米技术可以操纵受体组织，并导致受体激活和随后的信号转导过程发生变化。
