FIGURE 3:

WAVE recruits VASP for enhanced actin-based motility in vivo. (a) Lifeact-GFP imaging (WT WAVE and ΔPRD-WAVE) or Lifeact-mCherry imaging (ΔEVH1-VASP) of ventral enclosure in embryos with reintroduced wild-type WAVE or with mutant WAVE and VASP lacking putative interaction sites. Going from left to right, images are shown just before, at the moment of, and just after leader cell touch. Right, zooms of the boxed red areas. Reintroduced wild-type WAVE looks normal (see Figure 2), but introduction of either of the mutants gives leader cell protrusions that are blunted and only slightly in advance of adjacent pocket cells, as if VASP is not present (Figure 2). z-stack projections over several micrometers. Spinning disk fluorescence microscopy; ventral view, anterior is to the left. See also Supplemental Videos S3, S4, and S6. These differences are confirmed by pocket area measurements (b) and leader cell speed measurements (c). ΔPRD-WAVE and ΔEVH1-VASP have significantly smaller pocket sizes than reintroduced wild-type (p < 0.0001 and p = 0.049, respectively) and slower leader cell motility (p = 0.0015 and 0.01, respectively), although pocket cell speeds are unchanged with respect to wild type. (d) Synthetic lethal assay with embryonic survival represented as percentage of total eggs laid. On RNAi against WASP, most VASP-null embryos do not survive. ΔEVH1-VASP and ΔPRD-WAVE have much reduced survival compared with reintroduced wild-type proteins (p < 0.0001 for both), although both mutants are about as viable as reintroduced wild-type in absence of RNAi treatment (unpublished data). All data are represented as averages ± SD. p values calculated with the Student's t test. Bar, 15 μm; zoom, 7.5 μm.