Quantitative Imaging of Lateral ErbB1 Receptor Signal Propagation in the Plasma Membrane Peter J. Verveer, Fred S. Wouters, Andrew R. Reynolds, and Philippe I. H. Bastiaens

Supplementary Material

Supplemental Figure 1. EGF does not leak off the beads. Shown are three cells expressing ErbB1-GFP that were exposed to beads for 1 min. Only the cell that was in contact with a bead shows whole-cell phosphorylation of the receptor. The other two are within the same radius from the bead as the phosphorylated cell but do not show any response. Note the bead in the upper right corner that is close to the upper cell without being in contact with it. This indicates that bead-cell contact is essential for the phosphorylation response, and therefore, leakage of EGF from the bead is not significant.

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Supplemental Figure 2. Comparison of expression levels of ErbB1-GFP in MCF7 cells with that of endogeneous ErbB1 in A431 cells. Cells were starved for 4 hours and incubated with a saturating amount of fluorescently labeled Cy3-EGF (0.2 mg/ml) for 1 min, washed with ice-cold PBS, and fixed. (A) Fluorescence intensities of ErbB1-GFP versus fluorescence intensities of bound Cy3-EGF in MCF7 cells (n = 80) demonstrating that total Cy3 fluorescence is proportional to expression of ErbB1-GFP and that non-specific binding of Cy3-EGF to cells is negligible. (B) Normalized histograms showing the distribution of ErbB1 expression levels in MCF7 cells (n = 63, black bars) versus A431 cells (n = 80, grey bars) measured by the fluorescence of bound Cy3-EGF. Average fluorescence intensities were 4.3 ± 0.9 10⁶ for A431 cells and 2.2 ± 1.2 10⁶ for MCF7 cells. If one assumes a typical value of 1 10⁶ ErbB1 receptors per A431 cell, the amount of ErB1 receptors in transfected MCF cells was 0.5 ± 0.25 10⁶, half that of A431 cells. For imaging experiments, cells were used at expression levels corresponding to approximately 2 to 6 10⁵ ErbB1 receptors.

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Supplemental Figure 3. Confocal sections of live MCF7 cells expressing ErbB1-GFP (green) before and after 30 min of stimulation with Cy3-labeled EGF-beads (red). Sectioning allows vesicular structures to be distinguished from plasma membrane associated structures. (A) Before stimulation, (B) after 30-min EGF-bead stimulation. Arrowheads indicate clear vesicular structures that appear inside the cell.

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Supplemental Figure 4. Phosphorylation response of ErbB3-GFP in the presence of a minor fraction of ErbB1. (A) Phosphorylation populations of ErbB3-GFP coexpressed with ErbB1 at a ratio of 10:1 after 5 min of stimulation with 0.1 mg/ml EGF. (B) Control experiment showing the phosphorylation populations of ErbB3-GFP alone after 5 min of stimulation with 0.1 mg/ml EGF. This experiment shows that ErbB1-ErbB3 dimers are transient and that a single ErbB1 molecule can phosphorylate multiple ErbB3 molecules.

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