Figure 2. .

Effect of R416W and R239C mutations on the de novo GFAP IF network formation in IF-free cells. SW13/cl.2 (A–C) and primary astrocytes derived from GFAP/vimentin-null mice (D-F) were transiently transfected with either wild-type (A and D), R416W (B and E), or R239C (C and F) GFAP. At 48 h after transfection, the distribution of GFAP was assessed by confocal immunofluorescence microscopy with use of the rabbit polyclonal anti-GFAP antibody. When expressed in SW13/cl.2 cells, wild-type GFAP assembled into bundled filaments that extended throughout the cytoplasm (A). In contrast, cells transfected with either R416W (B) or R239C (C) GFAP-expression plasmids exhibited only GFAP-positive aggregates. In the IF-free mouse astrocytes, wild-type GFAP assembled into extended filaments at the cell periphery with some perinuclear accumulations (D), whereas R416W mutant GFAP formed punctuate aggregates scattered throughout the cytoplasm without any detectable filaments (E). Expression of R239C GFAP also induced numerous GFAP aggregates in the cytoplasm (F). For both R416W and R239C GFAP, all the transfected cells had aggregates. Bars = 10 μm.