FIGURE 5.

The GBD of ForC is required for proper cellular localization. A, examples of ForC accumulation at cell-cell contacts (left) and macropinosomes (right) using a GFP-tagged ForC construct encompassing the GBD and FH3 regions (residues 1–385). B, dynamic behavior of GFP-ForC 1–385 ectopically expressed in wild type cells (see also supplemental Movie 1). Numbers indicate the time (s). C, schematic illustration of the used ForC constructs containing or lacking the GBD and FH3 domains in this study. The numbers indicate amino acid residues (top). Equal amounts of total cellular proteins from transformants expressing the indicated GFP-tagged constructs were loaded per lane, subjected to SDS-PAGE, blotted onto nitrocellulose, and labeled with anti-GFP mAb 264-449-2 followed by alkaline phosphatase-conjugated anti-mouse IgG (bottom). D, the GBD and FH3 regions are both indispensable for proper intracellular localization. The cells were fixed and labeled with polyclonal anti-GFP antibodies to visualize the GFP fusion proteins (green). Filamentous actin was stained with rhodamine phalloidin (red). In contrast to GFP-ForC 1–385, which was enriched in the crowns and colocalized with filamentous actin, the ForC constructs lacking either GDB or FH3 were entirely cytoplasmic and showed no specific colocalization with F-actin. Confocal sections are shown in A, B, and D. Scale bars, 10 μm.