Ribosome distribution in HeLa cells during the cell cycle

Abstract

In this study, we employed a surface-specific antibody against the large ribosome subunit to investigate the distribution of ribosomes in cells during the cell cycle. The antibody, anti-L7n, was raised against an expansion segment (ES) peptide from the large subunit ribosomal protein L7, and its ribosome-surface specificity was evident from the positive immuno-reactivity of ribosome particles and the detection of 60 S immune-complex formation by an immuno-electron microscopy. Using immunofluorescent staining, we have microscopically revealed that ribosomes are dispersed in the cytoplasm of cells throughout all phases of the cell cycle, except at the G2 phase where ribosomes show a tendency to gather toward the nuclear envelope. The finding in G2 cells was confirmed by electron microscopy using a morphometric assay and paired t test. Furthermore, further observations have shown that ribosomes are not distributed immune-fluorescently with nuclear envelope markers including the nuclear pore complex, the integral membrane protein gp210, the inner membrane protein lamin B2, and the endoplasm reticulum membrane during cell division we propose that the mechanism associated with ribosome segregation into daughter cells could be independent of the processes of disassembly and reassembly of the nuclear envelope.

Figure 1. Characterization of the surface-specific properties of anti-L7n antibody against ribosome particles.

(A) Immuno-reactivity of anti-L7n antibody against L7, L7ΔN54, and L7N54 analyzed by SDS-PAGE with Coomassie blue staining (I) and Western blotting (II). L7ΔN54 is a mutant of L7 that lacks the first 54 amino acid residues at the NH₂-region of L7; N54 is a peptide that consists of the first 54 amino acid residues of L7. (B) Dot blotting assay using anti-L7n antibody. Each dot contains one OD₂₅₄ unit of HeLa ribosome particles (80 S) or 50 µg of S100 fraction (C) Immuno-reactivity of anti-L7n antibody against the ribosome (TP80) and the cytosolic S100 fraction analyzed by SDS-PAGE with Coomassie blue staining (I) and Western blotting (II). Each lane was loaded with 50 µg each of TP80 or cytosolic S100. (D) A sucrose density gradient analysis of the 60 S dimers (marked by arrow) from the reaction mixture after incubation of the 60 S subunit with anti-L7n. (E) Electron microscopy images of the 60 S immune-complexes. Bar = 20 nm.

Figure 2. The ribosome distribution of synchronized HeLa cells during the cell cycle.

(A) Cytometry measurement of the synchronized HeLa cells. (B) The cellular distribution of the ribosomes within HeLa cells during cell division was detected using anti-L7n, and co-stained with MAb414 antibody, which is specific to the nuclear pore complex. (C) A serial confocal microscopic section to show the ribosome gathering toward to the nucleus in the G2 cell. The spacing between sections was 1 µm. The representing picture is a merged image of ribosome and MAb414 staining. Bar = 10 µm.

Figure 3. Morphometric analysis of the ribosome population in the G1 and G2 phases.

(A) Example of a higher magnification view of a G1 cell with a mounted triple-lattice grid for counting the density of ribosomes. (B) An expanded view shows how the grid was mounted on a region proximal to the nuclear envelope. (C) An enlarged view shows the exact co-localization of the ribosome particle on the center white dot of the grid that represents one positive score. NM, nuclear membrane; No, nucleolus.

Figure 4. Co-localization of ribosome particles with the nuclear regulator protein gp210.

(A) The cellular distribution of the ribosomes within the HeLa cells during cell division was detected using anti-L7n, and co-stained with anti-gp210 antibody, which is specific to the nuclear membrane. (B) A serial confocal microscopic section to show the ribosome gathering toward to the nucleus in the G2 cell. The spacing between sections was 1 µm. The representing picture is a merged image of ribosome and gp210 staining. Bar = 10 µm.

Figure 5. Co-localization of ribosome particles with the inner nuclear lamin B2.

(A) The cellular distribution of the ribosomes within the HeLa cells during cell division was detected using anti-L7n, and co-stained with anti-lamin B2 antibody, which is specific to the nuclear inner membrane. (B) A serial confocal microscopic section to show the ribosome gathering toward to the nucleus in the G2 cell. The spacing between sections was 1 µm. The representing picture is a merged image of ribosome and lamin B2 staining. Bar = 10 µm.

Figure 6. The ribosome distribution in HeLa cells under the p180 knock-down.

(A) siRNA knock-down of the expression of p180. (B) The cellular distribution of the ribosomes within HeLa cells that had been down-regulated with respect to expression of p180 during cell division as detected by anti-L7n and MAb414 antibodies. Bar = 10 µm. (C) The ratio determined from the fluorescent intensity in each paired daughter cell during late telophase, stained by anti-L7n antibody. Ratio of mock was obtained from the non-siRNA knock-down cells.