Transfection of dominant-negative ezrin construct into PBT cells ablates surface structures. PBT cells were “nucleofected” with either plasmid encoding ezrin FERM-GFP (A-C) or encoding GFP alone (D-F). After 24 hours of culture, portions of each preparation of cells were prepared for confocal fluorescence microscopy by fixation, permeabilization, and staining with Alexa Fluor 568 phalloidin. The remaining portions were prepared for conventional SEM using chemical drying with tetramethylsilane. FERM-transfected cells showed localization of the FERM-GFP protein (A) in submembranous regions, whereas GFP-transfected cells showed diffuse cytoplasmic and nuclear staining (D). GFP-transfected cells had a fine punctate distribution of actin (arrowheads), which is characteristic of normal PBT (E). In contrast, FERM-GFP–transfected cells generally lacked this fine punctate distribution of actin (B). SEM of PBT cells transfected with FERM-GFP revealed a distinctive “bald” phenotype (C). This “bald” phenotype was not observed in GFP-transfected cells (F). Bar indicates 5 μm.