Supplemental Materials

This article contains the following supporting material:

• Supplemental Movie 1 - Cdc42-null fibroblasts form normal filopodia and lamellipodia. Cdc42(-/-) cells were transfected with an expression vector encoding EGFP-Β-actin fusion protein resulting in fluorescent labelling of the actin cytoskeleton. Immunofluorescence time lapse microscopy revealed normal formation of filopodia and lamellipodia in these cells. (27 s/frame collection rate; 12 frames/s display rate; cf. Figure 4A). Scale bar = 5μm.
• Supplemental Movie 2 - Expression of dnCdc42 in Cdc42-null cells results in increased membrane blebbing and loss of trailing edge. Phase contrast time lapse microscopy of Cdc42(-/-+ N17) cells indicated membrane blebbing and loss of trailing edge of cells. Quantification of 4 different movies of Cdc42(fl/-), Cdc42(-/-) and Cdc42(-/-+ N17) revealed the differences in membrane blebbing described in the result section. (18 s/frame collection rate; 12 frame/s display rate; cf. Figure 4C). Scale bar = 50μm.
• Supplemental Movie 3 - Supplememtary Movies 3, 4 and 5.Unstable direction of cell migration in Cdc42(-/-+ N17) cells in a wound closure assay, but not in Cdc42-null cells. A wound closure migration assay of Cdc42(fl/-) (movie 3), Cdc42(-/-) (movie 4) and Cdc42(-/-+ N17) cells (movie 5) was monitored by phase contrast time lapse microscopy during 6h. The leading edge is stably oriented towards the scratch in Cdc42(fl/-), but also in Cdc42(-/-) cells. In Cdc42(-/-+ N17) cells the leading edge was not stably oriented towards the scratch, but pointed in all possible directions. (4 min/frame collection rate; 12 frames/s display rate; cf. Figure 5B). Scale bar = 100μm.
• Supplemental Movie 4
• Supplemental Movie 5
• Supplemental Movie 6 - Supplementary Movies 6 and 7. Filopodium formation in Cdc42(fl/-) and Cdc42(-/-) ES cells. Phase contrast time lapse microscopy showing filpodium formation in Cdc42(fl/-) (movie 6) and Cdc42(-/-) (movie 7) embryonic stem cells. (10 s/frame collection rate; 12 frames/s display rate; cf. Figure 4B). Scale bar = 10μm.
• Supplemental Movie 7
• Supplemental Figure 1 - Normal spreading kinetics in the absence of Cdc42. Spreading kinetics of Cdc42-null cells on plastic is similar to control cells (scale bar = 100μm).
• Supplemental Figure 2 - Quantification of serum or migration induced signalling. A) Western blots of starved confluent Cdc42(fl/-), Cdc42(-/-), and Cdc42(-/-+ N17) cells stimulated with serum for the indicated times. Quantification of chemiluminescence signals was performed by using a CCD camera (LAS 1000, Fujifilm) and the software programs Image Reader LAS 1000 V1.1 and Image Gauge V3.01 (Fujifilm). Western blots for tubulin were used for correction of different loading. Asterisks indicate significant increase of the signal (p<0.05) between timepoint 0 min and 5 min within a given cell line; whereas two asterisks indicate significant difference (p<0.05) between the induction after 5 min and 30 min (cf. Figure 1C). B) Western blots of signal transduction in migrating Cdc42(fl/-), Cdc42(-/-), and Cdc42(-/-+ N17) cells at different time points after wounding. Quantification of chemiluminescence signals was performed by using a CCD camera (LAS 1000, Fujifilm) and the software programs Image Reader LAS 1000 V1.1 and Image Gauge V3.01 (Fujifilm). Western blots for tubulin were used for correction of different loading. (cf. Figure 7B).
• Supplemental Figure 3 - Β-catenin localizes to the leading edge of migrating cells independently of Cdc42. ?nPolarized?nΒ-catenin localization at the leading edge in migrating Cdc42(fl/-), Cdc42(-/-) and Cdc42(-/-+ N17) cells 2 h after wounding (scale bar = 10μm).
• Supplemental Table 1 - Quantification of cell area, elongation ratio and tail length in fibroblastoid cells. Quantification of cell area, elongation ratio and tail length in Cdc42(fl/-) cells and both mutant fibroblastoid cell lines is shown. Quantification was performed using MetaMorph 6.0 software.