Reversible Dimerization of EGFR Revealed by Single-Molecule Fluorescence Imaging Using Quantum Dots
Reversible Dimerization of EGFR Revealed by Single-Molecule Fluorescence Imaging Using Quantum Dots
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DOI：
10.1002/chem.200902963
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发表时间：
2010-01-01
影响因子：
4.3
通讯作者：
Biju, Vasudevanpillai
中科院分区：
文献类型：
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作者：
Kawashima, Nagako;Nakayama, Kenichi;Biju, Vasudevanpillai
目前的工作探讨了表皮生长因子受体（EGFR）参与细胞信号传导的侧向传播的分子间相互作用。通过结合EGFR胞外结构域中的EGF配体激活EGFR，随后EGFR二聚化启动细胞信号传导。我们通过单分子显微镜、福斯特共振能量转移（FRET）和原子力显微镜研究了活细胞中EGFR之间的相互作用。通过分析量子点（QD）标记的EGFR单分子的时间相关强度和传播轨迹，我们发现EGFR的信号二聚体[（EGF-EGFR）（2）]通过异源二聚体[EGF（EGFR）（2）]的可逆缔合在细胞膜中连续形成。此外，我们发现EGFR激活的横向传播通过异源二聚体与前二聚体的瞬时缔合发生[（EGFR）（2）]。我们用荧光共振能量转移技术从量子点标记的异源二聚体到Cy 5标记的前二聚体以及相关的拓扑学和荧光成像证实了激活的EGFR和前二聚体之间的瞬时关联。在没有扩展的单分子荧光成像和通过使用生物缀合的QD的情况下，EGFR的侧向激活中的可逆受体二聚化仍然模糊。
The current work explores intermolecular interactions involved in the lateral propagation of cell-signaling by epidermal growth factor receptors (EGFRs). Activation of EGFRs by binding an EGF ligand in the extracellular domain of the EGFR and subsequent dimerization of the EGFR initiates cell-signaling. We investigated interactions between EGFRs in living cells by using single-molecule microscopy, Forster resonance energy transfer (FRET), and atomic force microscopy. By analyzing time-correlated intensity and propagation trajectories of quantum dot (QD)-labeled EGFR single molecules, we found that signaling dimers of EGFR [(EGF-EGFR)(2)] are continuously formed in cell membrane through reversible association of heterodimers [EGF(EGFR)(2)]. Also, we found that the lateral propagation of EGFR activation takes place through transient association of a heterodimer with predimers [(EGFR)(2)]. We varified the transient association between activated EGFR and predimers using FRET from QD-labeled heterodimers to Cy5-labeled predimers and correlated topography and fluorescence imaging. Without extended single-molecule fluorescence imaging and by using bio-conjugated QDs, reversible receptor dimerization in the lateral activation of EGFR remained obscured.