(A) Mammalian P bodies and stress granules are spherical. Quantification of the circularity of stress granules (SG) and P bodies (PB) in HeLa cells, which expressed GFP-tagged DCP1a (PB) or G3BP2 (SG) from BACs (Poser et al., 2008). Cells were stressed with 1 mM sodium arsenate for 1 hr. Because of surface tension, liquid-like structures are expected to display a near circular shape (Hyman et al., 2014). A perfect circle has a circularity of 1. See ‘Materials and methods’ for details about how the circularity was determined. The mean and the SD are shown (n=118 PB, n=165 SG). (B) P bodies and stress granules show fusion behavior. Time-lapse microscopy of P bodies (DCP1a-GFP) and stress granules (G3BP2-GFP) in arsenate-stressed HeLa cells. Stress granules undergo frequent fusions at the beginning of a stress stimulus and form large spherical structures in cells after extended exposure to stress. Scale bars: 1 µm. Also see corresponding Videos 14, 15. (C) The stress granule protein G3BP2 shows fast internal rearrangement within stress granules, consistent with a liquid-like state. Kymograph of a stress granule (G3BP2-GFP) induced through arsenate stress and an amyloid (Q103-GFP) aggregate in HeLa cells after a half-bleach event (Brangwynne et al., 2009). PolyQ aggregates are used as a control for a solid-like structure. Note that only stress granules but not polyQ assemblies show a redistribution of fluorescence from the bleached to the unbleached area (from left to right). (D) P bodies and stress granules but not solid-like polyQ aggregates are sensitive to hexanediol. Quantification of the effect of 3.5% 1,6-hexanediol treatment on stress granules and P bodies induced through arsenate stress, and Q103 aggregates. The normalized mean fluorescence intensities of the structures are plotted over the duration of the treatment. Error bars are SD (n=5 PolyQ, n=31 SG and n=22 PB). (E) Mammalian stress granules do not co-aggregate with misfolded proteins. HeLa cells expressing BAC-encoded G3BP2-GFP (SG) were transfected with a plasmid coding for mutant luciferase-mCherry. Formation of stress granules was induced by arsenate stress (2 hr), proteasome inhibition (10 µM MG132, 3 hr) or heat stress (43°C, 2 hr). Scale bar: 10 µm.