Figure 1.

Microinjection of WA domains causes a rapid reorganization of the actin cytoskeleton and alters Arp2/3 location in resting and stimulated cells. MTLn3 cells were microinjected with a WASP-WA or Scar-WA peptide/dextran mix or with dextran alone, and fixed and stained for F-actin (phalloidin) and Arp2/3 (p34 antibodies) at different times after injection (a-d, 10 min; e-h, 30 min; i-l, 60 min after injection), or after stimulation with 5 nM EGF for 3 min 30 min after injection (m-p). A. F-actin (1 and 3rd column) and Arp2/3 (2nd and 4^th column) distribution patterns: cells microinjected with the WA domains (WASP-WA peptide, left 2 columns; Scar-WA peptide, right 2 columns) accumulate F-actin in perinuclear region, while Arp2/3 is depleted from leading edges, even in stimulated cells. Arrows, injected cells; arrowheads, Arp2/3 complex at the leading edge; small double arrows, Arp2/3 complex at the leading edge in an injected cell where actin has already accumulated in the perinuclear region. Scale bar: 20μm. B. Quantification of cell area: the cell area was normalized to the area of control non-injected cells. WA-injected cells display significantly smaller area (P<0.001) than control cells. C. Quantification of total F-actin in microinjected cells: the integrated density value for the phalloidin fluorescence was used as a measure of total F-actin content in the cells, normalized to the levels in control non injected cells. An average of 30 cells (13-54) was measured for each time point. Both WASP-WA and Scar-WA injected cells display a significant increase in F-actin content 10 minutes after injection (* P<0.001 and P<0.005 respectively for Scar-WA and WASP-WA).