Human chromosomes 9, 12, and 15 contain the nucleation sites of stress-induced nuclear bodies

Abstract

We previously reported the identification of a novel nuclear compartment detectable in heat-shocked HeLa cells that we termed stress-induced Src-activated during mitosis nuclear body (SNB). This structure is the recruitment center for heat shock factor 1 and for a number of RNA processing factors, among a subset of Serine-Arginine splicing factors. In this article, we show that stress-induced SNBs are detectable in human but not in hamster cells. By means of hamster>human cell hybrids, we have identified three human chromosomes (9, 12, and 15) that are individually able to direct the formation of stress bodies in hamster cells. Similarly to stress-induced SNB, these bodies are sites of accumulation of hnRNP A1-interacting protein and heat shock factor 1, are usually associated to nucleoli, and consist of clusters of perichromatin granules. We show that the p13-q13 region of human chromosome 9 is sufficient to direct the formation of stress bodies in hamster>human cell hybrids. Fluorescence in situ hybridization experiments demonstrate that the pericentromeric heterochromatic q12 band of chromosome 9 and the centromeric regions of chromosomes 12 and 15 colocalize with stress-induced SNBs in human cells. Our data indicate that human chromosomes 9, 12, and 15 contain the nucleation sites of stress bodies in heat-shocked HeLa cells.

Figure 1

Western blotting analysis of hamster B14-150 cells with anti-HAP antibodies. (Left panel) Total extracts of human HeLa cells (H) hamster B14-150 cells (B) and hamster>human somatic cell hybrid HY-1916 were fractionated onto a 7.5% SDS-PAGE and analyzed in Western blotting with an immunopurified polyclonal antibody directed against the recombinant human HAP protein (Weighardt et al., 1999). HY-1916 cells were analyzed both before (−) and after (+) heat shock. (Right panel) To prove the antibody specificity, Western blotting analysis of B14-150 cells was also performed in the absence (−) or in the presence (+) of purified recombinant antigen (see “Materials and Methods”). The same filter was hybridized with the G.T.U-88 mAb to γ-tubulin as a control for protein loading. The antibody–antigen complex was revealed with a peroxidase-conjugated anti-rabbit antibody and the enhanced chemiluminescence system. Molecular mass markers are indicated.

Figure 2

The heat shock-induced redistribution of HAP in hamster cells requires specific human chromosomes. Human HeLa cells, hamster B14-150 recipient cells, and HY-8F6, HY-1916, and HY-75E1 multi-chromosomal hamster>human cell hybrids were fixed with formaldehyde and analyzed by indirect immunofluorescence with anti-HAP rabbit polyclonal antibodies. The antigen–antibody complex was revealed with a rhodamine-conjugated sheep anti-rabbit secondary antibody. HAP distribution was revealed by confocal laser microscopy both in unstressed (37°C) and in heat-shocked (42°C) cells.

Figure 3

Human chromosome 9 directs the recruitment of HAP and HSF1 to stress-induced SNBs in hamster cells. Unstressed (37°C) and heat-shocked (42°C) recipient B14-150 hamster cells and monochromosomal GM-10611A hamster>human cell hybrids containing HSA 9 were analyzed by indirect immunofluorescence with the anti-HSF1 10H8 mAb and the anti-HAP polyclonal antibody. The distribution of HSF1 and HAP proteins was revealed with FITC-conjugated goat anti-rat IgG antibody and rhodamine-conjugated goat anti-rabbit IgG antibody, respectively. Confocal laser images of the same GM-10611A cells stained with anti-HSF1 and anti-HAP antibodies are shown.

Figure 4

Ultrastructural analysis of stress bodies in human cells and hamster>human cell hybrids. HeLa cells and GM-10611A hamster>human hybrid were heat shocked at 42°C for 1 h and after 1 h of recovery at 37°C, were fixed with formaldehyde, stained with the EDTA technique, and analyzed in electron microscopy. The dense “core” of the stress bodies and the clustering of perichromatin granules are detectable in both cell lines. Scale bars, 0.1 μm.

Figure 5

Ideogram of HSA9. The different chromosomal fragments present in GM-10611A, RH-9L132, RH-9L159, and in MCH hamster>human cell hybrids are shown. On the right part of the figure are shown confocal laser microscopy images of MCH cells, either unstressed (37°C) or heat shocked (42°C), stained with anti-HAP antibodies and rhodamine-conjugated goat anti-rabbit IgG antibodies.

Figure 6

Stress-induced SNBs colocalize with pericentromeric heterochromatin of HSA9, and centromeric regions of HSA12 and 15. HeLa cells were heat shocked at 42°C for 1 h and were allowed to recover 1 h at 37°C. After fixation in formaldehyde, cells were hybridized to biotinylated probes specific for satellite III DNA of HSA9, and α-satellite sequences on chromosome X, HSA12, and HSA15 (see “Materials and Methods”). Cells were costained with anti-HAP polyclonal antibodies to detect stress-induced SNBs. The distribution of HAP and of the biotinylated probes was revealed by indirect immunofluorescence with a rhodamine-conjugated goat anti-rabbit antibody and with FITC-conjugated avidin (see “Materials and Methods” for details). Confocal laser microscopy images of hybridized cells were taken to reveal the distribution of HAP (in red) and of the biotinylated probe (green). Images were merged to detect colocalization between satellite DNA sequences and stress bodies. On the left, confocal laser microscopy images of four consecutive optical sections (I–IV, 0.5 μm in depth) of the same cell hybridized to a probe (pHuR98) that recognizes the pericentromeric heterochromatin of HSA9. On the right, images of cells hybridized to probes specific for α-satellite of HSA12, HSA15, and chromosome X.

Figure 7

Quantitative analysis of the colocalization between specific chromosomes and stress-induces SNBs. Heat-shocked HeLa cells were independently hybridized to biotinylated probes the for heterochromatic region of HSA9 (pHuR98), HSA12 (pBR12), HSA15 (pMC15), and chromosome X (pDMX1). Cells were costained with anti-HAP polyclonal antibodies to detect stress-induced SNBs. The distribution of HAP and of the biotinylated probe was revealed by indirect immunofluorescence with a rhodamine-conjugated goat anti-rabbit antibody and with FITC-conjugated avidin. Confocal laser microscopy images were taken, and for each chromosome, we counted the number hybridization signals colocalizing with stress bodies. (A) Colocalizing signals in cells with one or two stress-induced SNBs. We analyzed 68 cells (25 of which had a single body) stained for HSA9, 17 cells (8 with one body) stained for HSA12, 15 cells (7 with one body) stained for HSA15, and 16 cells (7 with one body) stained for chromosome X. (B) Cells with more than two stress-induced SNBs were analyzed as in A. We analyzed 211 cells for HSA9, 47 cell for HSA12, 43 cells for HSA15, and 80 cells for chromosome X. ▪, HSA9. ▴, HSA12. ●, HSA15. ○ and dashed line, chromosome X.