FIG. 5.

Localization of Abi-1 and Wave-1 to the leading edge of the lamellipodium is dependent on the WAB domain of Abi-1 and the Abi-binding region of Wave-1, respectively. (A) Wild-type MEFs were infected with retroviruses encoding GFP-tagged Abi-1 wild-type and mutant forms, and the Abi-1 proteins were localized by direct observation of the GFP signal (left panels). Actin staining is shown in red (middle panels), and GFP and actin signals are merged in the right panels. Cells were plated for 30 min on fibronectin-coated coverslips, fixed, and visualized by confocal microscopy. (B) Percentages of cells expressing the indicated GFP-Abi-1 forms that exhibit (black bars) or lack (white bars) GFP signal at the leading edge of the lamellipodium. (C) Abi-1 oligomerization depends on the Syntaxin-1-binding region. The indicated Flag-Abi-1 forms and GFP-Abi-1 were coexpressed in 293T cells, and the Flag-Abi-1 proteins were immunoprecipitated (IP) with anti-Flag antibody. Coimmunoprecipitated GFP-Abi-1 was detected with anti-GFP (upper panel). Whole-cell lysates were immunoblotted (IB) with GPF (middle panel) and Flag antibodies (lower panel). (D) Expression of Myc-tagged wild-type Wave-1 and Wave-1Δ34-92 in MEFs. MEFs were infected with retroviruses encoding the indicated Wave-1 proteins, and cell lysates were immunoblotted with anti-Myc antibody. (E) Localization of Wave-1 to the leading edge of the lamellipodium is dependent on the Abi-1-binding region of Wave-1. Wild-type MEFs were infected with a retrovirus encoding Myc-tagged Wave-1 wild type or Wave-1Δ34-92. Cells were plated for 2 h and 15 min on fibronectin-coated coverslips, fixed, and visualized by using anti-Myc antibody.