Addition of purified G3BP1 protein to a cell lysate induces LLPS to assemble G3BP1-positive granules. (A) Schematic illustrating the generation of lysate granules from U2OS cells stably expressing G3BP1-GFP. (B) Addition of G3BP1 purified protein to a cell lysate from G3BP1-GFP U2OS cells induces LLPS of G3BP1 in a dose-dependent manner when the concentration of G3BP1 is increased by 5 µM with purified protein. LLPS is strongly inhibited in the absence of lysate. Images were taken 30 min following formation of lysate granules. Scale bar, 10 µm. (C) Addition of BSA or hnRNPA1 does not induce LLPS in lysates from G3BP1-GFP U2OS cells. Scale bar, 10 µm. (D) Purified G3BP1 lacking the RNA-binding domain (ΔRBD) or dimerization domain (ΔNTF2L) is unable to induce LLPS with RNA (top), nor does it induce LLPS in U2OS cell lysates expressing G3BP1-GFP (bottom). Scale bar, 10 µm. (E) Both two-component LLPS of G3BP1 and RNA (top) as well as LLPS induced by increasing the concentration of G3BP1 by 20 µM in U2OS cell lysates expressing G3BP1-GFP (bottom) are inhibited by the nsP3 peptide (LTFGDFDE, nsP3-8 hereafter) from Chikungunya virus. Scale bar, 10 µm. (F) Liquid properties of G3BP1-positive granules induced by increasing the concentration of G3BP1 by 20 µM using purified G3BP1 in U2OS cell lysates expressing G3BP1-GFP. Granules grow and fuse over time. Arrows indicate a fusion event between granules. Scale bar, 5 µm. (G) FRAP performed on G3BP1-GFP within lysate granules induced by increasing concentrations of purified G3BP1 show that granules are mobile. Graph represents mean ± SD, n = 10 granules per condition. (H and I) Brightfield phase diagram (H) with varying G3BP1 and lysate concentrations 60 min after induction (scale bar, 20 µm) and pictogram (I) indicating whether droplet formation was observed for indicated increases in G3BP1 concentration and lysate concentrations.
Figure 1.

Addition of purified G3BP1 protein to a cell lysate induces LLPS to assemble G3BP1-positive granules. (A) Schematic illustrating the generation of lysate granules from U2OS cells stably expressing G3BP1-GFP. (B) Addition of G3BP1 purified protein to a cell lysate from G3BP1-GFP U2OS cells induces LLPS of G3BP1 in a dose-dependent manner when the concentration of G3BP1 is increased by 5 µM with purified protein. LLPS is strongly inhibited in the absence of lysate. Images were taken 30 min following formation of lysate granules. Scale bar, 10 µm. (C) Addition of BSA or hnRNPA1 does not induce LLPS in lysates from G3BP1-GFP U2OS cells. Scale bar, 10 µm. (D) Purified G3BP1 lacking the RNA-binding domain (ΔRBD) or dimerization domain (ΔNTF2L) is unable to induce LLPS with RNA (top), nor does it induce LLPS in U2OS cell lysates expressing G3BP1-GFP (bottom). Scale bar, 10 µm. (E) Both two-component LLPS of G3BP1 and RNA (top) as well as LLPS induced by increasing the concentration of G3BP1 by 20 µM in U2OS cell lysates expressing G3BP1-GFP (bottom) are inhibited by the nsP3 peptide (LTFGDFDE, nsP3-8 hereafter) from Chikungunya virus. Scale bar, 10 µm. (F) Liquid properties of G3BP1-positive granules induced by increasing the concentration of G3BP1 by 20 µM using purified G3BP1 in U2OS cell lysates expressing G3BP1-GFP. Granules grow and fuse over time. Arrows indicate a fusion event between granules. Scale bar, 5 µm. (G) FRAP performed on G3BP1-GFP within lysate granules induced by increasing concentrations of purified G3BP1 show that granules are mobile. Graph represents mean ± SD, n = 10 granules per condition. (H and I) Brightfield phase diagram (H) with varying G3BP1 and lysate concentrations 60 min after induction (scale bar, 20 µm) and pictogram (I) indicating whether droplet formation was observed for indicated increases in G3BP1 concentration and lysate concentrations.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal