WAVE2 is required for lamellipodial protrusion in fibrinogen-adherent MKs. (A) Time-lapse record of spreading of fibrinogen-adherent ES cell–derived WAVE2^+/− and WAVE2^−/− MKs in the presence of 100 nM phorbol myristate acetate (PMA). A WAVE2^−/− MK displays enhanced dorsal ruffling and peripheral filopodial projection (arrowheads), while WAVE2^+/− MKs transiently shows filopodia within 5 minutes after adhesion (arrows) and lamellipodial formation thereafter. Scale bar equals 20 μm. (B) MKs on day 12 were plated onto fibrinogen for 60 minutes in the absence of an agonist (top 2 cells). In another set, MKs were plated for 120 minutes in the absence or presence of 100 nM PMA (bottom 5 cells). Cells were fixed, permeabilized, and stained with rhodamine-conjugated phalloidin to mark F-actin (red) and with anti-WAVE1, antivinculin, or anti-Arp3 antibodies followed by Alexa 488 (green), and analyzed by confocal microscopy (see “Image acquisition for platelet and MK spreading” for complete image acquisition information). Scale bar equals 20 μm. Arrows indicate peripheral normal lamellipodia formation. Arrowheads indicate filopodia-dominant morphology. (C) A total of 50 cells (WAVE2^+/−, □; or WAVE2^−/−, ■) were analyzed for attachment area and for the number of cells with lamellipodia-dominant morphology after 120 minutes incubation in the presence of PMA. Data are means (± SD).