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. 2005 Mar;25(5):1680–1695. doi: 10.1128/MCB.25.5.1680-1695.2005

FIG. 4.

FIG. 4.

N-WASP associates with WT Cdc42 in an intracellular vesicular compartment. (A) Cells were microinjected with plasmids encoding GFP-N-WASP (WT or H211D non-GTPase binding mutant) and HA-tagged WT or N17 inactive mutant Cdc42 (plasmid ratio, 1:2), incubated for 4 h at 37°C, fixed, and stained with a Cy3-conjugated anti-HA IgG Fab fragment. Multiphoton donor FLIM was undertaken to determine the extent of FRET between GFP-N-WASP (donor) and anti-HA-Cy3 (acceptor). FRET population determination was possible only with the WT GFP-N-WASP/WT Cdc42 pair; biexponential decay kinetics were not observed with either the H211D non-GTPase binding GFP-N-WASP or the N17 inactive mutant of Cdc42 labeled with anti-HA Fab-Cy3. Where appropriate, the subcellular localization of maximal τ decrease and highest FRET population is indicated by a white arrow. Red arrows indicate the accumulation of nuclear GFP-N-WASP in the presence of a coexpressed or N17 Cdc42 or the GTPase-binding-deficient GFP-N-WASP variant. High-resolution imaging of GFP-N-WASP/WT Cdc42 demonstrates interaction in vesicles (bottom panels) but no change in the GFP lifetime of the cell protrusion-targeted subpopulation of N-WASP (as indicated by red arrows). (B) FRET efficiency histogram compiled from all the data sets (n = 6) for each N-WASP plasmid combination. FRET efficiency = 1 − τda/τd, where τda is the pixel-by-pixel fluorescence lifetime of the donor in the presence of acceptor and τd is the average lifetime of the donor in the absence of acceptor (unstained controls). (C) Coimmunoprecipitation of the N-WASP-Cdc42 complex. In panel I, cells were transfected with various constructs as indicated and lysed. Western blotting was carried out on transfected versus nontransfected cell extracts, and membranes were blotted for total Cdc42 to allow a comparison between transfected and endogenous Cdc42 levels. In panel II, cells were transfected with GFP-N-WASP and lysed. Lysates were immunoprecipitated using either a control IgG or anti-GFP antibody. A fraction (one-fifth) of unbound material (S) was run alongside the antibody-bound protein (P) and Western blotted for GFP and Cdc42 protein.