FRαTb cells expressing GFP-Cdc42 (B and G), GFP-N-WASP (C), actin–GFP (D), GFP-CRIB (E) or GFP-Cdc42-L61 (F) were pulsed with TMR-dextran (R-Dex; A–F) or A568-Tf (G) or A488-Tf (A) for 20 s at 37°C and rapidly washed, fixed and imaged under TIRF illumination (depth ∼100 nm). (H) Histogram shows the extent of co-localization of endosomes labelled with TMR-dextran (R-Dex) or A-Tf (TfR) with expressed GFP-tagged constructs, GFP-Cdc42 (WT), GFP-N-WASP (N-WASP), actin–GFP (actin), GFP-CRIB (CRIB) or GFP-Cdc42-L61 (L61). Extent of non-specific co-localization using the procedures in Materials and Methods is ∼3%. Data were obtained from 10–15 cells, threee to five experiments and represented as wt. mean ± SEM. Scale bar, 10 μm.