Figure 5.
(A) Localization of GFP-WASP in Dictyostelium cells (WASPhypo) migrating toward a cAMP gradient. For examining GFP-WASP, cells were pulsed for 4.5 h at 6-min intervals. Fluorescence images were taken from live aggregation-competent cells migrating toward a chemoattractant gradient. GFP-WASP localizes to the leading edge and the uropod, which is recapitulated by GFP-B-GBD. GFP-WASP variant lacking WH1-B domains do not show prominent localization because GFP-WH1 shows uniform distribution. Arrows indicate the direction of gradient. Bar, 5 μm. (B) Localization of GFP-WASP in WASPTK cells. GFP-WASP still shows biased distribution at the leading edge and uropod, but appears to be associated with vesicular structures. Higher expression of GFP-WASP showed more diffuse distribution. (C) Localization of YFP-WASP to the leading edge. CFP-Coronin and YFP-WASP were coexpressed in wasphypo cells, and their localizations in chemotaxing cells were examined in time-lapse recording of alternate exposures (1 s) for CFP and YFP. Images were acquired and analyzed with multidimensional acquisition utility of Metamorph (Universal Imaging) software. Coronin localizes to sites of dynamic actin assembly and functions as a reporter for F-actin distribution in live cells.