Figure 1

Mutant-FUS expression delays the assembly and expedites the disassembly of stress granules in human cells. (A) Representative fluorescence images of HEK-293 cells expressing GFP-FUS (WT, R495X or H517Q) upon treatment with 0.25 mM sodium arsenite for 0, 40 and 90 min. The extent of stress granule formation in each line is illustrated with low magnification (40x) images using the anti-G3BP (red) stress granule marker (columns 1, 3 and 5). The localization of GFP-FUS (green) with respect to the nuclei (DAPI; blue) and stress granules is demonstrated within high magnification (100x) images (columns 2, 4 and 6). Scale bar = 20 and 5 μm, respectively, in low and high magnification images. (B) Quantification of the percentage of cells with stress granules at 40 min for the lines in (A) that are either induced (+) or not induced (−) to express GFP-FUS reveal that expression of GFP-FUS R495X causes a significant decrease in the number of cells with stress granules compared to controls. All error bars represent SEMs. Statistically significant differences were determined by one-way ANOVA and Tukey’s post-hoc test (****P < 0.0001, *P < 0.05). Additional significant comparisons include, but are not shown for clarity: uninduced GFP-FUS WT versus induced GFP-FUS R495X (P <0.001), uninduced GFP-FUS H517Q versus induced GFP-FUS R495X (P <0.001), and induced GFP-FUS H517Q versus induced GFP-FUS R495X (P < 0.01). (C) Western blot and densitometry analyses of cell lines in (B) and naive HEK-293 cells (UT) reveal that exogenous (exo) GFP-FUS proteins are expressed at levels similar to each other and within 2-fold of endogenous (endo) FUS (exo/endo FUS ratio). (D) Stress granule disassembly was assessed as described in (B) for GFP-FUS WT, R495X and H517Q lines after stress was removed for 90 min. Significantly fewer GFP-FUS R495X expressing cells contain stress granules.