Functional Las17-gfp remains localized independently of actin. (A) Localization of Las17p at 25°C after treatment of cells with latrunculin-A. Strain YMW173G homozygous for LAS17:GFP was grown in liquid YPD to log phase at 25°C and incubated with latrunculin-A (100 μM final concentration) in DMSO or an equivalent volume of DMSO. Images show cells fixed after 30 min exposure to latrunculin-A, which were briefly fixed and then stained with Alexa-phalloidin. Bar, 5 μm. (B) Cells were grown as in A, fixed for 1 h, and then digested with zymolyase to make spheroplasts. Spheroplasts were labeled with rabbit anti-GFP IgG (left panels) and mouse monoclonal anti-actin IgG (right panels), which were revealed by goat anti-rabbit FITC and goat anti-mouse Cyanine 3 secondary antibodies using appropriate filters. No overlap between filter channels was detected in control exposures.