1. Lateral Propagation of EGF Signaling after Local Stimulation Is Dependent on Receptor Density 
Asako Sawano, Shuichi Takayama, Michiyuki Matsuda, Atsushi Miyawaki 
Developmental Cell 
Volume 3, Issue 2, Pages (August 2002)
DOI: /S (02)

2. Figure 1 Visualization of Rhodamine-EGF-EGFR Complexes
Time-lapsed images of A431 cells (A and B), normal COS cells (C and D), and COS cells transfected with the cDNA for EGFR (E and F) when incubated with 50 ng/ml rhodamine-labeled EGF. The fluorescence signals were monitored every 10 s through the rhodamine channel. Images taken after 10 s (A, C, and E) and 20 min (B, D, and F) of incubation are presented. For estimation of EGFR plasma membrane expression level, images (C) and (E) were taken under the same optical conditions and are displayed using the same grayscale. In contrast, image (A) was captured with 20-fold-less sensitivity.
(E) EGFR-overproducing COS cells and untransfected COS cells are indicated by asterisks and arrowheads, respectively. Scale bar, 10 μm.
Developmental Cell 2002 3, DOI: ( /S (02) )

3. Figure 2 Microfluidic Channel for Laminar Flow Experiments
(A) Top view of the capillary network.
(B) A bright-field micrograph showing the junction where the three inlets converge. The channel contained confluent COS cells. A 20× objective lens was used.
(C) Fluorescence micrograph of the same field as in (B). The FITC-EGF and rhodamine-EGF fluorescence images were captured separately and merged. Scale bar, 100 μm.
(D) Dose-response curves for EGF-induced Ras activation in normal COS cells expressing Raichu-Ras. The EGF-induced increase in the emission ratio of Raichu-Ras (R/R0; the relative difference of the observed ratio [R] and the prestimulus value [R0]) was measured in culture dish (dotted line) and in the microfluidic channel (solid line). The saturating level of R/R0 ranged from 1.2 to 1.4. Each point is the mean ± SD of three to six experiments.
Developmental Cell 2002 3, DOI: ( /S (02) )

4. Figure 3
Local Stimulation of Normal COS Cells with Rhodamine-EGF for Visualization of Ras Activation
(A) Fluorescence image of Raichu-Ras before stimulation. The region of rhodamine-EGF exposure is shaded in violet. Across the laminar flows, four ROIs are assigned. Scale bar, 10 μm.
(B) Rhodamine-EGF fluorescence after 10 min stimulation. The outline of the COS cell is shown by a dotted line.
(C) Superimposed outlines of the cell at 0 (black) and 20 (red) min.
(D) A series of dual-emission ratio (535 ± 12.5 nm to 480 ± 15 nm) images presented in intensity-modified display (IMD) mode. Time points (min) after initiation of stimulation are shown in the top right corners.
(E) Fluorescence image of Raichu-Ras in another normal COS cell before stimulation with rhodamine-EGF (violet). Four ROIs are assigned as in (A). Scale bar, 10 μm.
(F) Rhodamine-EGF fluorescence after 20 min stimulation in the COS cell shown in (E), which is outlined by a dotted line. The lens was focused to see the punctate fluorescence pattern characteristic of endocytosis.
(G) A series of dual-emission ratio images from the COS cell shown in (E), presented in a manner similar to that in (D).
(H and I) Temporal profiles of the ratios in the ROIs indicated in (A) and (E), respectively. The changes in ratio were normalized to the initial ratio values. Red circles and squares are the data points from the stimulated side: ROIs 1 and 2, respectively. Blue circles and squares are from the unstimulated side: ROIs 3 and 4, respectively.
Developmental Cell 2002 3, DOI: ( /S (02) )

5. Figure 4
Local Stimulation of a Normal COS Cell with Rhodamine-EGF for Visualization of Tyrosine Phosphorylation beneath the Plasma Membrane
(A) Fluorescence image of Picchu-X with four ROIs; the rhodamine-EGF flow is shaded in violet. Scale bar, 10 μm.
(B) Rhodamine-EGF fluorescence images after 10 and 20 min stimulation, with the outline of the COS cell depicted by a dotted line. Over the monolayer culture is a round cell, which was intensely labeled (indicated by an asterisk).
(C) A series of dual-emission ratio images presented in IMD mode. Time points (min) after the start of stimulation are shown in the top right corners.
(D) Temporal profiles of the ratios in the ROIs indicated in (A). Data points are plotted as in Figures 3H and 3I.
(E) At 30 min, a ratio image was captured after global stimulation with rhodamine-EGF.
Developmental Cell 2002 3, DOI: ( /S (02) )

6. Figure 5
Local Stimulation of EGFR-Overproducing COS Cells with Rhodamine-EGF for Visualization of Ras Activation
(A) Fluorescence image of Raichu-Ras in a COS cell transfected with EGFR cDNA. The cell was nearly in contact with the left wall of the capillary. Four ROIs and the rhodamine-EGF flow (violet) are shown. Scale bar, 10 μm.
(B) Rhodamine-EGF fluorescence after 10 min stimulation. The dotted line depicts the outline of the COS cell. A neighboring cell, which produced a large quantity of EGFRs, is indicated by an arrowhead. An arrow defines other neighboring cells that were not transfected. This image was taken with 10-fold-less sensitivity than the images in Figures 3B and 4B.
(C) A series of dual-emission ratio images presented in IMD mode. Time points (min) after initiation of stimulation are shown in the top right corners.
(D) Fluorescence image of Raichu-Ras in another EGFR-overexpressing COS cell before stimulation with rhodamine-EGF (violet). Four ROIs are assigned as in (A). Scale bar, 10 μm.
(E) Rhodamine-EGF fluorescence after 10 min stimulation in the COS cell shown in (D), which is outlined by a dotted line.
(F) A series of dual-emission ratio images from the COS cell shown in (D), presented in a manner similar to that in (C).
(G and H) Temporal profiles of the ratios in the ROIs indicated in (A) and (D), respectively. Data points are plotted as in Figures 3H and 3I. Subsequent addition of 50 ng/ml rhodamine-EGF is indicated by an arrowhead in (H).
Developmental Cell 2002 3, DOI: ( /S (02) )

7. Figure 6
Local Stimulation of an EGFR-Overproducing COS Cell with Rhodamine-EGF for Visualization of Tyrosine Phosphorylation beneath the Plasma Membrane
(A) Fluorescence image of Picchu-X with four ROIs, with the rhodamine-EGF flow shaded in violet. Scale bar, 10 μm.
(B) Rhodamine-EGF fluorescence images after 10 and 20 min stimulation. The outline of the COS cell is demarcated by a dotted line. The camera settings were the same as those for the image in Figure 5B.
(C) A series of dual-emission ratio images presented in IMD mode. Time points (min) after initiation of stimulation are shown in the top right corners.
(D) Temporal profiles of the ratios in the ROIs indicated in (A). Data points are plotted as in Figures 3H and 3I.
Developmental Cell 2002 3, DOI: ( /S (02) )

8. Figure 7
Propagation of Ras Activation after Blockade of Endocytosis and Visualization of Rap1 Activation in Normal COS Cells
(A) Fluorescence image of Raichu-Ras in a COS cell after treatment with MDC but before stimulation. This cell was neighbored by a few other cells on the right side, as indicated by arrowheads (C). The rhodamine-EGF flow (violet) is shown. Scale bar, 10 μm.
(B) Rhodamine-EGF fluorescence images after 10 and 20 min stimulation. The outline of the COS cell is shown by a dotted line.
(C) A series of dual-emission ratio images for Ras activation presented in IMD mode. Time points (min) after initiation of stimulation are shown in the top right corners. After 20 min, no activation of Ras was observed in the neighboring cells (arrowheads), which had also been treated with MDC.
(D) Fluorescence image of Raichu-Rap1 in a COS cell with the rhodamine-EGF flow shaded in violet. Scale bar, 10 μm.
(E) Rhodamine-EGF fluorescence images captured after 10 and 20 min stimulation. The outline of the COS cell is shown by a dotted line.
(F) A series of dual-emission ratio images of Rap1 activation presented in IMD mode. Time points (min) after initiation of stimulation are shown in the top right corners.
Developmental Cell 2002 3, DOI: ( /S (02) )