Figure 3.

Microinjection of Scar-WA domain triggers a complete reorganisation of barbed ends in resting cells, but does not prevent a further increase in barbed ends following EGF stimulation. MTLn3 cells were starved for 3 hours and microinjected with Scar-WA domain 30 minutes prior to barbed end labelling. Barbed ends were visualised by permeabilising the cells in presence of 0.45 uM labelled monomeric actin, and quantified as detailed in Material and Methods. A. Barbed ends staining before (a, d) and 30 sec (b, e) or 1 min (c, f) after EGF stimulation. In resting cells (a), barbed ends are localised at the edge of existing lamellipods (arrowheads), in focal contacts (arrows) and in a perinuclear diffuse pattern. Stimulation with EGF triggers a large increase in barbed ends specifically at the leading edge (arrowheads). Cells injected with Scar-WA domain display a diffuse and strong cytosplamic barbed end pattern (d), with an enrichment at focal contacts (arrows) after stimulation but not at the leading edge (e, f). Scale bar =10μm. B, C. Quantification of barbed ends at the leading edge (B) and total barbed ends in the cells (C). The barbed ends were measured locally as the mean fluorescence within 1.1 um at the leading edge (B), or globally as the integrated density of the fluorescence over the total cell area (C). Both numbers were normalised to control uninjected cells within the Scar-WA unstimulated sample (EGF 0). White bars, control cells; black bars, Scar-WA injected cells. The numbers in bar represents the number of cells analysed. Stars: significantly different from control (*, P<0.05; **, P<0.01).