FIG. 5.

VASP is required for the effect of SH2-Bβ on actin-based motility. (A) WT or ΔActA6 Listeria cells were incubated in Xenopus egg extract supplemented with either GST or GST-SH2-Bβ. The movement of individual bacteria was measured, and velocities were calculated. Bars represent means plus SEM. *, P < 0.05 compared to extracts supplemented with GST. (B) ActA-coated beads were incubated in motility medium, with or without 150 nM VASP and 1 μM of the indicated proteins. For each assay, the movement of n beads was measured, and the average velocity was calculated. From top to bottom, n = 5, 6, 6, and 4 without VASP and n = 7, 7, 8, and 8 with VASP. Bars represent means plus standard deviations (SD). (C) Time-lapse images of ActA-coated bead movement in motility medium, with or without 150 nM VASP and/or 1 μM SH2-Bβ, as indicated. The large arrows refer to the initial position of the bead, while small arrows indicate the moving bead. Flows in the samples sometimes induced a drift of the objects. Bar, 40 μm. (D) The velocity of ActA-coated beads was determined in motility medium supplemented with increasing concentrations of VASP in the presence (solid circles) or absence (open circles) of 1 μM SH2-Bβ. Bars represent means ± SD, calculated for a set of n beads in each sample. For increasing concentrations of VASP, n = 7, 5, 8, 6, 8, and 11 with SH2-Bβ and n = 5, 5, 9, 8, and 10 without SH2-Bβ. (E) The velocity of N-WASP-coated beads was determined in motility medium supplemented with increasing concentrations of SH2-Bβ in the presence (open circles) or absence (solid circles) of 150 nM VASP. The bead movement was independent of VASP and SH2-Bβ. Bars represent means ± SD, calculated for a set of n beads. For increasing concentrations of SH2-Bβ, n = 10, 13, 14, and 13 without VASP and n = 9, 6, 5, and 7 with VASP.