Protein composition of G3BP1-induced lysate granules resembles the protein composition of stress granules in cells. (A) Primary antibodies conjugated to Alexa Fluor 647 secondary antibody were used to visualize proteins known to be stress granule constituents (left) and proteins known to be absent from stress granules (right). Scale bar, 10 µm. (B) Endogenous canonical stress granule constituents (TDP-43, ATXN2L, TIA1) were fluorescently tagged using CRISPR-Cas9 KI in U2OS cells. Endogenous TDP-43, ATXN2L, and TIA1 all localized to lysate granules. GFP from U2OS cells stably expressing GFP did not localize to lysate granules as visualized with anti-G3BP1 staining. Scale bars, 10 µm. (C) Overexpression of GFP-tagged stress granule constituents (FUS, hnRNPA1, or caprin 1) by transfection resulted in larger granules, demonstrating positive cooperativity. Lysates were seeded at equal concentrations. Images show granules 2 h after induction by increasing the G3BP1 concentration by 20 µM and associated quantification of average granule size at 2 h. Scale bar, 10 µm. Graph represents mean ± SD, n = 5 independent representative fields. ANOVA with Dunnett’s test was used to calculate P values for control versus FUS (***, P = 0.0002), hnRNPA1 (****, P < 0.0001), and caprin 1 (****, P < 0.0001). (D) Gene ontology analysis of proteins found to be enriched within G3BP1-induced lysate granules by mass spectrometry reveals that lysate granules share common features with cellular RNP granules. (E) Most proteins identified within lysate granules appear within the RNP Granule Database (http://rnagranuledb.lunenfeld.ca/) as Tier 1–4 proteins, indicating that lysate granules share common components with stress granules. (F–I) FRAP performed on G3BP1-GFP and GFP-ATXN2L shows similar mobility between stress granules within U2OS cells induced with 0.5 mM NaAsO2 (F and G) and lysate granules induced by increasing G3BP1 concentration by 20 µM using purified G3BP1 (H and I). Arrowheads indicate stress granules or lysate granules on which FRAP was performed. All images were acquired 30 min after induction. Scale bars, 10 µm. Graphs in G and I represent mean ± SD, n = 10 or 11 granules for G3BP1-GFP and GFP-ATXN2L from either cells or lysate.
Figure 2.

Protein composition of G3BP1-induced lysate granules resembles the protein composition of stress granules in cells. (A) Primary antibodies conjugated to Alexa Fluor 647 secondary antibody were used to visualize proteins known to be stress granule constituents (left) and proteins known to be absent from stress granules (right). Scale bar, 10 µm. (B) Endogenous canonical stress granule constituents (TDP-43, ATXN2L, TIA1) were fluorescently tagged using CRISPR-Cas9 KI in U2OS cells. Endogenous TDP-43, ATXN2L, and TIA1 all localized to lysate granules. GFP from U2OS cells stably expressing GFP did not localize to lysate granules as visualized with anti-G3BP1 staining. Scale bars, 10 µm. (C) Overexpression of GFP-tagged stress granule constituents (FUS, hnRNPA1, or caprin 1) by transfection resulted in larger granules, demonstrating positive cooperativity. Lysates were seeded at equal concentrations. Images show granules 2 h after induction by increasing the G3BP1 concentration by 20 µM and associated quantification of average granule size at 2 h. Scale bar, 10 µm. Graph represents mean ± SD, n = 5 independent representative fields. ANOVA with Dunnett’s test was used to calculate P values for control versus FUS (***, P = 0.0002), hnRNPA1 (****, P < 0.0001), and caprin 1 (****, P < 0.0001). (D) Gene ontology analysis of proteins found to be enriched within G3BP1-induced lysate granules by mass spectrometry reveals that lysate granules share common features with cellular RNP granules. (E) Most proteins identified within lysate granules appear within the RNP Granule Database (http://rnagranuledb.lunenfeld.ca/) as Tier 1–4 proteins, indicating that lysate granules share common components with stress granules. (F–I) FRAP performed on G3BP1-GFP and GFP-ATXN2L shows similar mobility between stress granules within U2OS cells induced with 0.5 mM NaAsO2 (F and G) and lysate granules induced by increasing G3BP1 concentration by 20 µM using purified G3BP1 (H and I). Arrowheads indicate stress granules or lysate granules on which FRAP was performed. All images were acquired 30 min after induction. Scale bars, 10 µm. Graphs in G and I represent mean ± SD, n = 10 or 11 granules for G3BP1-GFP and GFP-ATXN2L from either cells or lysate.

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