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First published online 19 August 2008
doi: 10.1242/jcs.031823

Journal of Cell Science 121, 3002-3014 (2008)
Published by The Company of Biologists 2008
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Heat shock causes a decrease in polysomes and the appearance of stress granules in trypanosomes independently of eIF2{alpha} phosphorylation at Thr169

Susanne Kramer1, Rafael Queiroz2,3, Louise Ellis1, Helena Webb1, Jörg D. Hoheisel3, Christine Clayton2 and Mark Carrington1,*

1 Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, UK
2 ZMBH, Im Neuenheimer Feld 282, 69120 Heidelberg, Germany
3 Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 580, 69120 Heidelberg, Germany


Figure 1
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  		Fig. 1. Growth recovery following heat shock. Trypanosome cultures were incubated a 41°C for 60, 90 or 120 minutes. Growth was monitored after cultures were returned to 27°C. One representative growth curve from three experiments is shown.

 

Figure 2
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  		Fig. 2. Effect of heat shock on translation and polysomes. (A) Changes in translation during heat-shock treatment. Cells were labelled for 20-minute windows at 27°C and during a heat-shock time course followed by recovery at 27°C. An autoradiograph of the SDS-PAGE analysis is shown. Two polypeptides with unchanged synthesis are marked with solid arrows. Examples of proteins that have reduced synthesis after 5 hours recovery are marked with dashed arrows. The stained gel was used to control for equal loading (bottom). One lane of the gel has been removed. (B) Changes in polysomes during heat shock treatment. Absorbance profiles at 254 nm of sucrose density gradients of cells incubated at 41°C for 15 and 30 minutes. 50 mM EDTA was added to the cell lysate to dissociate polysomes. Representative results from several experiments are shown.

 

Figure 3
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  		Fig. 3. Changes in mRNA steady-state levels following heat shock. Cell cultures were transferred from 27°C to either 37°C or 41°C over a time course, and RNA was analysed by northern blotting and microarrays. All probes used in this work were specific (supplementary material Fig. S2A). (A) Northern blot probed for total mRNA using an antisense oligonucleotide recognising the spliced leader; the spliced leader RNA (SLRNA) is indicated. One of three replica gels is shown, all three were used for quantification; error bars indicate ± s.d. (B) Oligonucleotide microarray slides were probed with RNA from cells incubated at either 27°C or 41°C. The total dataset is shown as a plot of the intensities for each spot. (C) The filtered dataset from B. (D) Northern blots probed for PDI-2, actin and {alpha}- and β-tubulin. Data from one representative experiment out of several repeats are shown. (E) Northern blots probed for HSP70 (Tb11.01.3110) and HSP83 (Tb10.26.1080). One out of two replica gels is shown. Quantification from both replica gels is shown.

 

Figure 4
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  		Fig. 4. The decrease in mRNA steady-state levels is reversible. Trypanosome cultures were transferred from 27°C to 41°C and returned to 27°C after 1 hour. Northern blots were probed for total mRNA, actin, tubulin and PDI-2.

 

Figure 5
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  		Fig. 5. Effect of heat shock on transcription and mRNA processing. (A,B) Trypanosome cultures were heat-shocked at 41°C or treated with 2 µg/ml sinefungin at 27°C, 37°C or 41°C. The decrease in mRNAs caused by the different treatments was analysed by northern blots probed for total mRNA (A), actin, tubulin and PDI-2 (B). Tubulin mRNAs aberrantly spliced by sinefungin (McNally and Agabian, 1992Go) are indicated by arrows.

 

Figure 6
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  		Fig. 6. Characterisation of T. brucei heat-shock stress granules. (A) Homologues of stress granule markers were expressed as eYFP fusion proteins. Fluorescent images of unstressed (27°C) and heat-shocked (1 hour 41°C) cells are shown. (B) The effect of heat shock on cells that express PABP1-eYFP from a modified endogenous locus; treatments are indicated. (C) Disappearance of heat-shock stress granules after a return to 27°C in cells that express PABP1-eYFP from a modified endogenous locus. The percentage of cells with visible granules was determined during recovery and the average values (n=100) from three slides is shown, error bars indicate ± s.d. (D) Optical sections through T. brucei cells that express PAPB1-eYFP or eIF4E3-eYFP after 60 minutes at 41°C are shown as projection (left) and some selected single plane images (right). (E) Colocalisation of eIF3B-eYFP and eIF4E3-mChFP to heat-shock stress granules.

 

Figure 7
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  		Fig. 7. Effect of cycloheximide on polysomes, stress granules and mRNA degradation during heat shock. (A) Cycloheximide prevents polysome dissociation and partially restores polysomes during heat shock. Polysomes were analysed in cells incubated (from top to bottom) at: 27°C; 41°C for 60 minutes; 27°C with cycloheximide for 30 minutes; 27°C with cycloheximide for 30 minutes, followed by 60 minutes at 41°C; 41°C for 60 minutes followed by 30 minutes at 41°C with cycloheximide and; 27°C for 30 minutes with puromycin. (B) Cycloheximide prevents formation of heat-shock stress granules. Fluorescent images of cells that express PABP1-eYFP were incubated (from top to bottom) at: 27°C; at 41°C for 60 minutes; 27°C with cycloheximide for 60 minutes; 27°C with cycloheximide for 60 minutes followed by 60 minutes at 41°C; 41°C for 60 minutes followed by 60 minutes with cycloheximide and; 27°C with puromycin for 60 minutes. (C) Cycloheximide prevents the heat-shock-induced decay for PDI-2 and Tubulin. Cultures were incubated at 41°C (left lanes), 27°C with cycloheximide (middle lanes), and 27°C with cycloheximide followed by incubation at 41°C (right lanes). Analyses of the northern blot for PDI-2 and tubulin are shown below.

 

Figure 8
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  		Fig. 8. T. brucei P-bodies. (A) T. brucei cells have in average 2.9 P-bodies. Projection of optical sections through puromycin-treated cells that express SCD6-eYFP. Similar Z-stacks (supplementary material Fig. S5A) were used to determine the number of P-bodies per cell: the average was 2.9 per cell (n=125) with little variation in different cell-cycle stages: 2.7 in 1K1N (n=95), 3.3 in 2K1N (n=17) and 3.3 in 2K2N (n=12) cells. (B) P-body components DHH1, SCD6 and XRNA. eYFP-DHH1 and SCD6-mChFP (left panel) or mChFP-DHH1 and XRNA-eYFP (right panel) were expressed in the same cell line from endogenous loci. Representative fluorescent images of untreated, and cells after 1 hour with puromycin, cycloheximide or heat shock (41°C) are shown. Solid arrows indicate the posterior pole of the cell. An enlargement of a heat-shocked cells expressing mChFP-DHH1 and XRNA-eYFP is shown to indicate the colocalisation of DHH1 and XRNA in all spots (dashed arrows), except at the posterior pole. (C) P-bodies and heat-shock stress granules. mChFP-DHH1 and PABP1-eYFP (left) or mChFP-SCD6 and PABP1-eYFP (right) were expressed in the same cell line from their endogenous loci. Representative fluorescent images of one untreated cell and several images of cells that have been heat shocked (1 hour 41°C) are shown. Dashed arrows point at granules that contain both a P-body marker (DHH1 or SCD6) and the stress granule marker PABP1, either colocalised or close to each other.

 

Figure 9
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  		Fig. 9. Formation of heat-shock stress granules is independent on eIF2{alpha} phosphorylation at T169. Heat shock treatment (1 hour 41°C) of cells expressing PABP1-eYFP from the endogenous locus and with either a single wild-type (T169T) or mutated (T169A) eIF2A gene (supplementary material Fig. S6). (A) Fluorescent images of two clonal cell populations expressing wild-type eIF2A (T2 and T3) or mutant eIF2A (A1 and A2) are shown. (B) Polysome analysis of the eIF2A T169A/– cells incubated at either 27°C or 41°C for 60 minutes.

 

Figure 10
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  		Fig. 10. Model of compartments that may determine mRNA fate in growing cells (left) and in heat-shocked cells (right).

 

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© The Company of Biologists Ltd 2008