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Figures
- Fig. 1.
Ultrastructure of arsenite-induced stress granules in human cells. HeLa (A,B) and HEK293 (C) cells were treated (B,C) or not (A) with arsenite for 30 minutes, as indicated. Cells were then processed for immunofluorescence with anti-eIF3 antibody (left panels) or for electron microscopy (middle panels). Electron microscopic images of glutaraldehyde-fixed, Epon-embedded cells, stained with lead citrate and uranyl acetate are shown. Cytoplasmic granular zones present only in stressed cells are indicated by a black arrow. Pm, plasma membrane; M, mitochondrion; Nu, nucleus. A region including a stress granule and the neighbouring cytosplasm is enlarged in the right panel of B and C, and a region of cytoplasm at the same magnification is shown in the right panel of A. Black arrowheads point to polyribosomes present in the HeLa control cells. The fine granular structure of the cytoplasmic granules in arsenite-treated cells is indicated by white arrowheads. Scale bars: 10 μm, 1 μm and 200 nm for left, middle and right panels, respectively.
- Fig. 2.
Accumulation of TIA1-GFP in arsenite-induced stress granules in HEK293 cells. HEK293 cells transfected with TIA1-GFP were treated with arsenite, fixed in glutaraldehyde and embedded in Lowicryl K4M. Thin sections were reacted with a primary anti-GFP antibody and a secondary antibody coupled to 10 nm gold particles. Gold particles are concentrated over a cytoplasmic granule (black arrows) whereas the surrounding cytoplasm, including mitochondria, is weakly labelled. Pm, plasma membrane; NE, nuclear envelope; M, mitochondrion; Nu, nucleus. Uranyl acetate staining. Scale bar: 500 nm.
- Fig. 3.
Ultrastructure of heat shock-induced stress granules in HeLa cells. HeLa cells were incubated at 42°C for 30 minutes and processed for immunofluorescence with anti-eIF3 antibody (left panel) or for electron microscopy (right panel). A heat shock-induced stress granule (black arrow) closely apposed to mitochondria (M) is shown. Note dense fibrillar patches (white arrowhead) within the stress granule. Scale bar: 1 μm.
- Fig. 4.
Ultrastructure of TIA1-induced stress granules in HEK293 cells. (A) Thin section of a TIA1-GFP transfected HEK293 cell fixed in glutaraldehyde and embedded in Epon. Large cytoplasmic granules are conspicuous (black arrows). Note electron-dense fibrillar patches (white arrowheads) within these granules. V, vacuole; M, mitochondrion. Scale bar: 1 μm. (B) Detection of the TIA1-GFP fusion protein with an anti-GFP antibody in paraformaldehyde-fixed, Lowicryl-embedded HEK293 cells. Two cytoplasmic granules (black arrows) are highly labelled with gold particles particularly concentrated over the dense fibrillar patches (white arrowheads). The surrounding cytoplasm is also significantly labelled, indicating a high level of expression of the TIA1-GFP fusion protein in this particular cell. M, mitochondrion; V, vacuole; Pm, plasma membrane. Scale bar: 500 nm. (C) Continuous stress granule remodelling in live cells. TIA1-GFP transfected cells were observed by fluorescence microscopy for 90 minutes. The region indicated by an arrowhead is enlarged below, and shows a piece of stress granule transferred from one granule to another. Scale bars: 2 μm.
- Fig. 5.
RNA composition of arsenite-induced stress granules analysed by high resolution in situ hybridization in HeLa cells. HeLa cells were treated with arsenite for 30 minutes and processed for electron microscopy. Specific RNA species were hybridized with biotinylated DNA probes, as indicated, and revealed by immunodetection with an antibiotin antibody coupled to gold particles. As illustrated, the 28S rRNA is low in the stress granule indicated by black arrows (A), whereas the 18S rRNA (B) and the poly(A) RNA (C) are highly enriched. Notice that, despite high intensity of labelling, the extracellular resin is devoid of gold particles. Pm, plasma membrane. Scale bar: 500 nm.
- Fig. 6.
Comparison of P-body ultrastructure in control and arsenite-treated HeLa cells. Control HeLa cells (A) and cells treated with arsenite for 30 minutes (B) were processed for immunofluorescence (left panels) or for immunoelectron (middle and right panels) microscopy with anti-p54 antibody. Endogenous Rck/p54 was identified in thin sections of Lowicryl embedded cells using a secondary antibody coupled to 10 nm gold particles. Pm, plasma membrane; Nu, nucleus. The induction of P-bodies by arsenite is visible in the left panel of B. In the middle panels, Rck/p54 can be seen to accumulate in roundish fibrillar structures with a diameter of 300-400 nm, consistent with the size of P-bodies detected by immunofluorescence. An enlarged view of a P-body is shown in the right panel, revealing tangentially cut 10-15 nm fibres indicated by arrowheads. Scale bars: 10 μm, 500 nm and 100 nm for left, middle and right panels, respectively.
- Fig. 7.
Ultrastructural characterization of P-bodies in arsenite-treated HeLa cells. HeLa cells were treated with arsenite for 30 minutes and processed for immunofluorescence (A) or electron microscopy (B-E). (A) Stress granules and P-bodies were detected with anti-eIF3 (red) and anti-p54 (green) antibodies, respectively. P-bodies are frequently associated with stress granules, as indicate by the arrowhead and enlarged in the insert. (B) Endogenous Rck/p54 was detected in thin sections of Lowicryl embedded cells, as in Fig. 6. (C) Thin section of an Epon-embedded cell, as in Fig. 1. Both B and C show a P-body (white arrowhead) contacting a stress granule (black arrowhead). (D) Enlargement of a P-body embedded in Epon reveals 10-15 nm (black arrowheads) and 4 nm (white arrowheads) fibres. The region encompassing the 10-15 nm and 4 nm fibres marked by a star is further enlarged in E. Scale bars: 500 nm (B,C); 100 nm (D,E).
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