Bioinformatic Analysis of Actin-Binding Proteins in the Nucleolus During Heat Shock

Figures

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  Figure 1. Heat shock induces nAC-GFP assembly in the nucleolus. HeLa cells were transfected with the nAC-GFP expression vector and treated with heat shock (42 °C for 60 min). Confocal images of nAC-GFP (green), FBL (red), NPM1 (magenta), and DNA (blue). Scale bar = 10 μm.

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  Figure 2. The reversibility of heat shock-induced nAC-GFP assembly is suppressed by the inhibition of Hsp70. HeLa cells were transfected with nAC-GFP expression vector and then treated with heat shock (42 °C for 60 min), followed by recovery at 37 °C. (a) The nAC-GFP assembly was observed over 60 min by confocal microscopy with time-lapse imaging. Confocal images of nAC-GFP (green) and DIC (grey) are shown. Scale bar = 10 μm. (b) Hsp70 inhibitor VER-155008 (VER; 50 μM) was added to the culture medium immediately before recovery at 37 °C. After 90 min of incubation at 37 °C, the cells were fixed. Confocal images of nAC-GFP (green) and DAPI (blue) are shown. Scale bar = 20 μm.

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  Figure 3. Prediction of actin-binding proteins localized to the nucleolus under heat shock conditions. (a) The workflow of bioinformatic analysis in this study. The datasets of actin-binding proteins and nucleolus localized proteins were filtered from UniProtKB and a nucleolus gene set of LOCATE-curated protein localization annotations (Harmonizome 3.0), respectively. In this study, proteomic data (LS-MS/MS) of human melanoma M14 cells under heat shock conditions were obtained from gene expression omnibus (GEO) and re-profiled. Protein 3D structures were predicted using AlphaFold2and AlphaFold-Multimer. (b) The Venn diagram illustrates the overlap of actin-binding proteins (2981 proteins, blue) and nucleolus-localized proteins (648 proteins, green). From this set, the gene names of 47 proteins common to both actin-binding and nucleolus-localized proteins were identified. Additionally, the Venn diagram includes proteins whose expression levels increased by more than 1.5-fold upon heat shock treatment (278 proteins, orange). (c) The IDRs in 47 proteins extracted by (b) were analyzed by using IUPred2A. The high disorder regions in this study were defined as amino acid region in which disorder…

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  Figure 4. Prediction of IDRs and three-dimensional structures of PDLIM7 and TRIOBP. The left graph indicates plots the accurate IDR predictions for PDLIM7, TRIOBP-1, TRIOBP-4, and TRIOBP-5, as determined by IUPred2A. The X- and Y-axes represent the position of the peptide chain in the protein and the disorder score, which indicates the degree of IDR, respectively. Regions with a disorder score of 0.5 or above indicate a high probability of being disordered regions. The right panels show the three-dimensional structure of the proteins predicted using AlphaFold2 and AlphaFold-Multimer. The color scale shows the predicted local distance difference test (pLDDT) score, which is a measure of prediction accuracy for each amino acid residue.