The Rap1 V12 mutant induces remodeling of cortical actin.

(A) Untreated K562 cells expressing YFP-Rap1 wt displayed cortical actin, which did not colocalize significantly with Rap1. (B) TPA-treated K562 cells expressing YFP-Rap1 wt show loss of cortical actin with apparent colocalization of actin and Rap1. (C) Untreated K562 cells expressing YFP-Rap1 V12 show loss of cortical actin and apparent colocalization of actin and Rap1. (D) TPA-treated K562 cells expressing YFP-Rap1 V12 show absent cortical actin with apparent colocalization of actin and Rap1. These findings were similar in 2 different experiments using 2 independently isolated K562-YFP-Rap1 V12 clones. K562 stable transfectants with or without TPA were grown 48 hours on glass coverslips, followed by fixation and staining for actin with rhodamine-conjugated phalloidin. YFP fusions and rhodamine-phalloidin were detected simultaneously by 2-color confocal imaging at a total magnification × 520.