Transcriptome, proteome, and protein synthesis within the intracellular cytomatrix

Abstract

Despite the knowledge that protein translation and various metabolic reactions that create and sustain cellular life occur in the cytoplasm, the structural organization within the cytoplasm remains unclear. Recent models indicate that cytoplasm contains viscous fluid and elastic solid phases. We separated these viscous fluid and solid elastic compartments, which we call the cytosol and cytomatrix, respectively. The distinctive composition of the cytomatrix included structural proteins, ribosomes, and metabolome enzymes. High-throughput analysis revealed unique biosynthetic pathways within the cytomatrix. Enrichment of biosynthetic pathways in the cytomatrix indicated the presence of immobilized biocatalysis. Enzymatic immobilization and segregation can surmount spatial impediments, and the local pathway segregation may form cytoplasmic organelles. Protein translation was reprogrammed within the cytomatrix under the restriction of protein synthesis by drug treatment. The cytosol and cytomatrix are an elaborately interconnected network that promotes operational flexibility in healthy cells and the survival of malignant cells.

Keywords: Cell biology; Molecular biology; Proteomics; Transcriptomics.

Graphical abstract

Figure 1

Illustration of cytomatrix isolation The cytosol and cytomatrix were sequentially extracted from HCT-15 cells. The cytosol was extracted with nonionic detergent containing mild solvent, whereas the cytomatrix was separated from the nuclear fraction using a buffered, stringent solvent. (A) Immunoblots of marker proteins in fractions. (B) Immunofluorescence image of keratin localization in cytosol-removed cells. (C) Immunofluorescence images of SUN2 (red) and Nesprin-1 (green) localization in HeLa cells. (D) Nesprin-1 is predominantly localized around the nucleus in SUN2 knockdown cells. (E-J) TEM micrographs of HCT-15 cells. Ultrastructure of the nuclear periphery of (E and F) an intact cell and (G-I) a cytosol-removed cell. (J) cytosol and cytomatrix removed core nuclei. (K) Protein expression in the cytosol versus cytomatrix of three cell lines. The cytosol and cytomatrix were run side by side, but bands were cut based on cell line to show differences in protein expression in corresponding compartments. Western blots were run in the same gel. The three cell lines show consistent results for predominantly cytosolic proteins (upper panel), equally distributed proteins (middle panel), and predominantly cytomatrix proteins (bottom panel). Cyt – cytosol, CMX – cytomatrix, M− mitochondria, INM – inner nuclear membrane, ONM – outer nuclear membranes, PM – plasma membrane carcass, PNS – perinuclear space.

Figure 2

Analysis of protein synthesis in the cytosol and cytomatrix (A) Protein distribution in the cytosol and cytomatrix of three cell lines under the normal growth conditions (left panel) and 500 nM of pp242 treatment for 24 h (right panel). (B) The effect of increasing concentration of motor protein inhibitors on protein distribution in the cytomatrix and cytosol of HCT-15 cells. Inhibitors of the dynein and kinesin proteins showed no effect on protein distribution. To minimize mixing during loading, the equal amounts of samples were run separately. Side by side running gel data for the same samples are presented in Figures S3A and S3B. (C and D) Polysome profile of the cytomatrix and cytosol of HCT-15 cells. (E) The incorporation of radiolabeled L-[³H]-phenylalanine into proteins of the cytosol, and the CMX in untreated and pp242-treated HCT-15 cells. L-[³H]-phenylalanine was added, and cells were incubated for 1 h. ∗p-value of one-way ANOVA, p < 0.0001. Data are given as means ± SE, n = 5. (F) eIF4E3 is localized in the cytomatrix of HCT-15 cells. (G) The pp242 increased eIF4E3 protein expression in the CMX of HCT-15 cells. (H) eIF4E-BP1 phosphorylation in the cytosol and cytomatrix with and without pp242 treatment of HCT-15 cells. (I) Hypothetical diagram of inverse relationship of the eIF4E1 and eIF4E3. eIF4E – eukaryotic initiation factor 4E. BP1 – eIF4E binding protein 1.

Figure 3

Mass spectrometry proteome analysis of the cytomatrix and cytosol of HCT-15 cells (A) Heatmap of protein expression from cytosol and cytomatrix of the control and pp242 treated HCT-15 cells. (B) Heatmap of 25 selected proteins in each differential analysis. (C and D) KEGG and Reactome pathway analyses of CMX proteome of the untreated and pp242 treated HCT-15 cells for 24 h. NES – normalized enrichment score.

Figure 4

Analysis of transcription and translation in the cytomatrix and cytosol of HCT-15 cells (A) Heatmap of transcripts from RNA-seq. (B) Heatmap of footprints from Ribo-seq. (C) Venn diagrams of the overlaps and unique upregulated and downregulated transcript sets obtained from bulk RNA-seq and Ribosome footprint. (D) Reactome set visualization of the enriched upregulated and downregulated pathways. (E and F) KEGG analysis of enriched pathways identified through Ribo-seq.

Figure 5

Upregulated pathways of the cytosol and cytomatrix of HCT-15 cells identified through Ribo-seq (A and B) Categorized cytosolic GOBP pathways. (C and D) Categorized cytomatrix GOBP pathways.