JCS02551 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 - Fig.S1. TIRF illumination can distinguish between dorsal and ventral surfaces of the lamellipodium. (a and b) Fixed keratocytes were incubated with 0,17 mm fluorescent beads and imaged using TIRF, epi-fluorescence, and DIC. After RGB colour combination of the three images, beads imaged by both TIRF and epi appear white whereas beads imaged by epi only appear red.

• Movie 1 - Movie 1. Keratocyte motility is both rapid and persistent. The lamellipodium remains flat on the substrate at all times. Cell body transport matches the rate of leading edge protrusion so that cells appear to glide over the substrate.

• Movie 2 - Movie 2. Lateral diffusion of DiIC12 is blocked at the leading edge. Epi-fluorescent images (upper left) show the spread of dye in the dorsal and ventral plasma membranes. TIRF images (upper right) show the spread of dye in the ventral membrane only. Merged image (lower left) shows epi in green and TIRF in red. Intensity scan (lower right) through the horizontal blue line graphically represents dye spreading. Dye reaches the leading edge via the dorsal surface at 11.29 seconds, but does spread to the ventral surface. Dye reaches the leading edge via the ventral surface at 17.71 seconds. Note that in sequential TIRF images, dye always fills from the back of the cell toward the front; the leading edge never becomes brighter than the lamellipodium interior. For each image pair the TIRF image preceded the epi image by 750 milliseconds.

• Movie 3 - Movie 3. Dye accumulation at the leading edge on the dorsal cell surface. Same sequence as SupVid-02 shown here at full time resolution (1.5 frame/second) between 8 and 34 seconds.

• Movie 4 - Movie 4. Keratocyte immobilized by CA treatment exhibiting centripetal flow within the lamellipodium.

• Movie 5 - Movie 5. Lateral diffusion of DiIC12 is not blocked at the leading edge of CA/CB treated cells. Panel arrangement same as SupVid-02. Dye reaches leading edge via dorsal surface at 8.51 seconds. At 10.62 seconds (next frame) dye appears at leading edge on the ventral surface. The double peak intensity profile clearly indicates that dye has spread from the dorsal to ventral cell membrane at both the front and back of the cell.

• Movie 6 - Movie 6. Epi-fluorescent time series of motile keratocyte homogenously labeled with Alexa-488 conjugated cholera toxin. There is no enrichment of the lipid ordered phase marker at the leading edge.