Reversible dimerization of EGFR revealed by single-molecule fluorescence imaging using quantum dots.
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, Förster 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.
Kawashima
Nagako
N
Health Technology Research Center, National Institute of Advanced Industrial, Science and Technology (AIST), 2217-14 Hayashi-Cho, Takamatsu, Kagawa 761-0395, Japan.
Nakayama
Kenichi
K
Itoh
Kohji
K
Itoh
Tamitake
T
Ishikawa
Mitsuru
M
Biju
Vasudevanpillai
V
D045744:Cell Line, Tumor; D019281:Dimerization; D066246:ErbB Receptors / Q000737:chemistry* / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D008024:Ligands; D018625:Microscopy, Atomic Force; D011485:Protein Binding; D045663:Quantum Dots; D015398:Signal Transduction
D005456:Fluorescent Dyes; D008024:Ligands; D066246:ErbB Receptors