Colocalization of pyrin with actin by confocal microscopy.

HeLa cells were transfected with pEGFP-MEFV.N plasmid. After fixing, cells were stained with phalloidin–Alexa Fluor 568. (A) GFP fluorescence showing staining of ruffles over the nucleus and lamellar structures (arrows). (B) Phalloidin staining of actin fibers. Shorter fibers are ruffles over the nucleus, and in lamellar structures (arrows). (C) Merged green and red channels with yellow signal at overlap. Arrows highlight overlap at nuclear and lamellar ruffles. (D) Colocalization mask computed using linear gating (see “Materials and methods”). (E-G) HeLa cells transfected with pEGFP-MEFV.N plasmid were treated with 10 μM cytochalasin D for 2 minutes, then fixed and stained with phalloidin–Alexa Fluor 568. GFP-pyrin (E) remains colocalized with the disintegrating actin scaffolding (F). Merged fluorescence images (G) demonstrate punctate colocalization overlying and surrounding the nuclear region. Magnification × 1000.