Supplemental Material

This article contains the following supporting material:

• Fig1HeLa - Activation of RhoA in a migrating HeLa cell. HeLa cells expressing Raichu-RhoA were imaged as described in the legend to Figure 1A.
• Fig1MDCK - Activation of RhoA in a migrating MDCK cell. MDCK cells expressing Raichu-RhoA were imaged as described in the legend to Figure 1B.
• Fig2RhoA - Video image of RhoA activity in EGF-stimulated Cos1 cells. Cos1 cells were transfected with pRaichu-RhoA and were imaged as described in the legend to Figure 2.
• SupFig1HeLa
• SupFig2RhoA
• SupFig5 - Video image of EGF-stimulated Cos1 cells expressing a dominant negative mDia1 mutant. Cos1 cells expressing Kaede-mDia1 ΔFH2 mutant were recorded for Kaede and differential interference contrast (DIC) images every 2 min for 60 min.
• Supplemental Figure 1 - Imaging of RhoA activities in migrating HeLa cells with Raichu-RhoA fused to the authentic carboxy-terminal region of RhoA. HeLa cells expressing Raichu-RhoA/RhoACT were imaged as described in the legend to Figure 1. Video image is also available (SupFig1HeLa.mov).
• Supplemental Figure 2 - Spatio-temporal activity of RhoA after stimulation with growth factor. HeLa cells expressing Raichu-RhoA/RhoACT were imaged as described in the legend to Figure 2. Video image is also available (SupFig2RhoA.mov).
• Supplemental Figure 3 - Rac1 does not require RhoA for the induction of lamellipodia. HeLa cells with or without expression vectors, indicated at the top of each panel, were stained with Alexa488-conjugated phalloidin. The lower panels show the higher magnification of the boxed region. Bars indicate 10 μm.
• Supplemental Figure 4 - Retention of mDia mutants at the membrane ruffles. Cells expressing KAEDE-mDia1 ΔN3 and KAEDE-mDia1 ΔN3 (HindIII) were used for the analysis. At time point 0, KAEDE proteins in the white-circled regions were photo-converted from green to red by laser irradiation. (A) Cells were imaged for Green-KAEDE, Red-KAEDE, and DIC every 5 sec. Pseudo-color Ratio images (Red-KAEDE / Green-KAEDE) were also created to demonstrate the retention of photo-converted KAEDE proteins. Representative images created after photoconversion are shown. (B) The intensities of Green-KAEDE and Red-KAEDE and Ratio values (Red/Green) along the white-dotted line are plotted against the distance from the edge of the cells.
• Supplemental Figure 5 - Retention of mDia at the membrane ruffles depends on active RhoA. (A) Cells expressing Dronpa-mDia1, Dronpa-mDia1 ΔN3 and Dronpa-mDia1 ΔN3 (HindIII) were used for the analysis. Fluorescence was erased to the background levels with a strong 488 nm laser (middle panels). Dronpa proteins at the membrane ruffles and the other peripheral region of cells shown with white-dotted lines were photo-activated by LD laser irradiation (in lower panels). Bars indicate 10 μm. (B) Time courses of the decrease of Dronpa fluorescence at the irradiated area. Diffusion of Dronpa proteins photoactivated at time point 1 was monitored for 15 seconds.