Figure 3

GFP-FUS R495X is weakly bound to stress granules and alters binding of stress granule-associated proteins. (A) Live cell images of GFP-FUS (WT and R495X) expressing HEK-293 cells transfected with mRFP-G3BP. Images are shown before (−) and after (+) treatment with 0.2 mM sodium arsenite (SA) for 1 hr. Scale bar = 10 μm. (B) Top three panels: exemplar GFP and mRFP images of a SA treated cell for a mRFP-TIA-1 FRAP experiment before and after photobleaching. The mRFP signal, but not GFP signal, is lost from the stress granule (indicated by arrow). Scale bar = 5 μm. Bottom four panels: fluorescence intensity profiles corresponding to the above panels (rotated 90° clockwise). (C) The recovery curve for GFP-FUS R495X in untransfected (UT; green triangle) cells are indicative of fast fluorescence recovery. The GFP-FUS R495X profile does not change upon transfection with either mRFP-G3BP (blue circle) or mRFP-TIA-1 (red square). (D) Nearly identical mobile fractions support the conclusions in (C). (E & F) Recovery curves for mRFP-G3BP (E) and mRFP-TIA-1 (F) differ for GFP-FUS WT (blue circle) and R495X (red square) expressing cells. (G) Mobile fractions for the curves in (E & F) are significantly higher for GFP-FUS R495X (black bars) relative to GFP-FUS WT (white bars) cells. Mobile fractions for mRFP-TIA-1 are the same for the following control experiments: GFP-FUS WT expressing cells (white bars), uninduced (UI) GFP-FUS WT cells (grey bar) and uninduced GFP-FUS R495X cells (hatched bar). Asterisks indicate statistically significant differences between cell lines as determined by two-way ANOVA and Tukey’s post-hoc test (****P < 0.0001) on data from at least n=2 independent experiments. All error bars represent SEMs. The total number (N) of stress granule analyzed is indicated on the recovery panels.