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Biology Articles » Methods & Techniques » Optical Bioimaging: From Living Tissue to a Single Molecule: Single-Molecule Visualization of Cell Signaling Processes of Epidermal Growth Factor Receptor » Figures

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- Optical Bioimaging: From Living Tissue to a Single Molecule: Single-Molecule Visualization of Cell Signaling Processes of Epidermal Growth Factor Receptor

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Fig. 1. Single-molecule visualization of Cy3-EGF using total internal reflection fluorescence microscopy (TIR-FM). A: Configuration of objective-type TIR-FM. By changing the incident angle of the laser beam, both apical and basal surfaces of the cell can be visualized (5, 6). B: Single-molecules of Cy3-EGF bound to EGFR on living cells. Cy3-EGF (0.2 ng/ml) was added to the culture medium of living A431 cells under a TIR-FM. Binding of Cy3-EGF was completely inhibited in the presence of 200 ng/ml of non-labeled EGF (inset). C: The fluorescence intensity change in single Cy3-EGF spots on living cells was plotted against time. The spots were photobleached in a single step (arrow). The duration between appearance (binding) and disappearance of Cy3-EGF depended on excitation intensity, indicating the disappearance was not due to dissociation. D: Distribution of the fluorescence intensity of Cy3-EGF and Cy3-anti-EGFR (EGFR1) on the surface of living cells. Histograms of the fluorescence intensity are fitted to a sum of two or three Gaussian distributions (lines). Position of the mean (arrow) and percentage fraction (number) of each Gaussian component are indicated. Numbers in parentheses are the percentage fractions expected from Poisson distributions with averages of 0.4 and 1.0, respectively. Modified from Ref. 5.

figure 1 

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Fig. 2. Dimerization of EGF receptors. A: Dimerization pathways of EGF/EGFR complexes were observed on living A431 cells in the presence of Cy3-EGF. Two different processes of dimerization, i.e., entrapment of EGF to pre-clusters of EGFR (upper row) and diffusion and collision of EGF/EGFR complexes (lower row) were observed. Most dimers (90%) were formed in the former pathway. B: Single-molecule FRET between Cy3-EGF and Cy5-EGF in a receptor dimer. A mixture of Cy3-EGF (0.2 ng/ml) and Cy5-EGF (0.8 ng/ml) was added to the culture medium of A431 cells. The specimen was excited with a green (532 nm) laser and fluorescence images of the same field in the Cy3-channel (565 – 595 nm) and Cy5-channel (650 – 690 nm) were acquired simultaneously by using dual view-optics. Time course of the fluorescence intensity change of a single spot was measured both in Cy3 and Cy5 channels and plotted against time after normalizing the peak intensity of each channel. Modified from Ref. 5.

 figure 2

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Fig. 3. Activation and signal transduction processes of EGFR. A: A431 cells were perforated by streptolysin O (SLO) and stimulated by Rh-EGF. Activation of EGFR was detected using a Fab' fragment against activated EGFR. The Fab' fragment was conjugated with a fluorophore, Alexa488. Rh-EGF (red) and Alexa488-Fab' (green) were observed simultaneously in single molecules. Positions of activated EGFR at EGF-binding sites are indicated (arrowheads). B: Recognition between activated EGFR and an adaptor protein Grb2. EGFR in the basal plasma membrane was activated using Cy5-EGF. Association and dissociation of single molecules of Grb2 conjugated with Cy3 at the N-terminus was observed at the position of Cy5-EGF. The off and on times of individual events can be determined form the change of fluorescence intensity over time (8).

 figure 3

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Fig. 4. Imaging of EGF-binding and intracellular calcium response in the same cells. Cells were stimulated with a 10-s pulse of Rh-EGF started at time 0. Number of EGF-binding on the surface of living cells was counted by changing the focus up and down (left). At the same cells, concentration of intracellular Ca2+ was measured using a fluorescence indicator Fluo-4 (right). Binding of EGF was observed 1 min after addition of EGF.

 figure 4

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