Addition of NPM1 to a cell lysate induces a distinct lysate granule that recapitulates the composition and properties of the nucleolus.(A) Addition of purified NPM1 protein to a cell lysate induces LLPS in a dose-dependent manner when the concentration of NPM1 is increased by more than 5 µM. Scale bar, 10 µm. (B and C) FRAP performed on cells or lysate from cells transiently expressing GFP-NPM1 shows similar mobility between the nucleolar NPM1 in intact U2OS cells and NPM1 in lysate granules induced by increasing the concentration of NPM1 by 15 µM using purified NPM1. White arrows indicate nucleolus or NPM1 lysate granule on which FRAP was performed. Nuclear boundary is indicated by yellow dashed line. All images were acquired 30 min after induction. Scale bar, 10 µm, n = 10 granules from either cells or lysate. Graph represents mean ± SD. (D) The nsP3-8 peptide from Chikungunya virus strongly inhibits the formation of G3BP1-induced lysate granules (top) but has no effect on the formation of NPM1-induced lysate granules (bottom). Scale bars, 10 µm. (E) Indicated primary antibodies were conjugated with Alexa Fluor 647 secondary, and either anti-G3BP1 or anti-NPM1 was conjugated with Alexa Fluor 488 secondary. Conjugated antibodies were added to U2OS lysates along with purified G3BP1 or NPM1. Scale bar, 10 µm. (F) Gene ontology (GO) analysis of proteins found to be enriched within NPM1-induced (increasing NPM1 concentration by 15 µM) lysate granules by mass spectrometry reveals that lysate granules show nucleolar features as well as some common features with G3BP1-induced (increasing G3BP1 concentration by 20 µM) lysate granules. MLO, membraneless organelle. (G) Venn diagrams showing the overlap of proteins identified through mass spectrometry between NPM1-induced lysate granules (increasing NPM1 concentration by 15 µM) and G3BP1-induced lysate granules (increasing G3BP1 concentration by 20 µM), as well as the overlap of proteins previously identified in stress granules (RNA Granule Database, see main text) and the nucleolar proteome as assessed by GO analysis of the PANTHER database. (H) G3BP1 lysate granule Z-scores (granule vs. lysate, x axis) were plotted against NPM1 lysate granule Z-scores (granule vs. lysate, y axis) to compare similarity in differentially expressed genes between G3BP1 and NPM1 lysate granules and were found to be highly dissimilar. Pearson’s correlation analysis (r = 0.195). (I) Heat map of the gene expression of the enriched RNAs (relative to their own control total cellular RNA or total lysate) in stress granules (Khong et al., 2017), G3BP1 lysate granules, and NPM1 lysate granules.
Figure 4.

Addition of NPM1 to a cell lysate induces a distinct lysate granule that recapitulates the composition and properties of the nucleolus. (A) Addition of purified NPM1 protein to a cell lysate induces LLPS in a dose-dependent manner when the concentration of NPM1 is increased by more than 5 µM. Scale bar, 10 µm. (B and C) FRAP performed on cells or lysate from cells transiently expressing GFP-NPM1 shows similar mobility between the nucleolar NPM1 in intact U2OS cells and NPM1 in lysate granules induced by increasing the concentration of NPM1 by 15 µM using purified NPM1. White arrows indicate nucleolus or NPM1 lysate granule on which FRAP was performed. Nuclear boundary is indicated by yellow dashed line. All images were acquired 30 min after induction. Scale bar, 10 µm, n = 10 granules from either cells or lysate. Graph represents mean ± SD. (D) The nsP3-8 peptide from Chikungunya virus strongly inhibits the formation of G3BP1-induced lysate granules (top) but has no effect on the formation of NPM1-induced lysate granules (bottom). Scale bars, 10 µm. (E) Indicated primary antibodies were conjugated with Alexa Fluor 647 secondary, and either anti-G3BP1 or anti-NPM1 was conjugated with Alexa Fluor 488 secondary. Conjugated antibodies were added to U2OS lysates along with purified G3BP1 or NPM1. Scale bar, 10 µm. (F) Gene ontology (GO) analysis of proteins found to be enriched within NPM1-induced (increasing NPM1 concentration by 15 µM) lysate granules by mass spectrometry reveals that lysate granules show nucleolar features as well as some common features with G3BP1-induced (increasing G3BP1 concentration by 20 µM) lysate granules. MLO, membraneless organelle. (G) Venn diagrams showing the overlap of proteins identified through mass spectrometry between NPM1-induced lysate granules (increasing NPM1 concentration by 15 µM) and G3BP1-induced lysate granules (increasing G3BP1 concentration by 20 µM), as well as the overlap of proteins previously identified in stress granules (RNA Granule Database, see main text) and the nucleolar proteome as assessed by GO analysis of the PANTHER database. (H) G3BP1 lysate granule Z-scores (granule vs. lysate, x axis) were plotted against NPM1 lysate granule Z-scores (granule vs. lysate, y axis) to compare similarity in differentially expressed genes between G3BP1 and NPM1 lysate granules and were found to be highly dissimilar. Pearson’s correlation analysis (r = 0.195). (I) Heat map of the gene expression of the enriched RNAs (relative to their own control total cellular RNA or total lysate) in stress granules (Khong et al., 2017), G3BP1 lysate granules, and NPM1 lysate granules.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal