FIGURE 4:

VASP's F-actin and profilin/G-actin binding activities are important for its effect on WAVE-based motility. (a) Lifeact-mCherry imaging of ventral enclosure in embryos carrying GFP-tagged VASP proteins mutant for profilin binding, F- and G-actin binding, and tetramerization (ΔPP, ΔFAB, ΔGAB, and ΔTET, respectively). Left image is just before leader cell touch, and right image is at the moment of contact. The leader cell protrusion is rounded and less in advance of the adjacent pocket cells in the ΔPP and ΔFAB cases as compared with the two others. (b) This gives correspondingly smaller pocket areas for ΔPP and ΔFAB (p = 0.016), whereas ΔGAB and ΔTET are identical to reintroduced wild-type protein (unpublished data; p = 0.79 and 0.87, respectively). See also Supplemental Videos S8 and S9. (c) Embryonic survival of mutant VASP embryos subjected to the synthetic lethal RNAi treatment. ΔTET had a level of survival like wild-type (Figure 3d), whereas ΔGAB was reduced (p = 0.04 as compared with reintroduced wild type), although not as much as ΔFAB and ΔPP, which were identical to the negative control EVH1 (p = 0.97 and 0.12, respectively). (d) PRD-VCA-WAVE–coated beads incubated in the motility mix with different forms of VASP. Left, representative comets at 10- to 15-min reaction time. See Figure 1 for pictures of wild-type, ΔEVH1-VASP, and no-addition comets. Phase contrast microscopy. Right, bead speeds normalized to the wild-type speed for each day, which was on average ∼0.8 μm/min. Two to four independent experiments were averaged for each condition. Wild type and no addition are replotted from Figure 1c for comparison. ΔTET-VASP addition is the same as wild type (p = 0.3), whereas ΔFAB-VASP gives identical speeds to no addition (p = 0.12). ΔGAB-VASP and ΔPP-VASP inhibit motility. All data are represented as averages ± SD. p values calculated with the Student's t test. Bars, 15 μm (a), 1 μm (d).