In trypanosomes there is an almost total reliance on posttranscriptional mechanisms to alter gene expression; here, heat shock was used to investigate the response to an environmental signal. Heat shock rapidly and reversibly induced a decrease in polysome abundance, and the consequent changes in mRNA metabolism were studied. Both heat shock and polysome dissociation were necessary for (1) a reduction in mRNA levels that was more rapid than normal turnover, (2) an increased number of P-body-like granules that contained DHH1, SCD6 and XRNA, (3) the formation of stress granules that remained largely separate from the P-body-like granules and localise to the periphery of the cell and, (4) an increase in the size of a novel focus located at the posterior pole of the cell that contain XRNA, but neither DHH1 nor SCD6. The response differed from mammalian cells in that neither the decrease in polysomes nor stress-granule formation required phosphorylation of eIF2α at the position homologous to that of serine 51 in mammalian eIF2α and in the occurrence of a novel XRNA-focus.Supplementary material available online at http://jcs.biologists.org/cgi/content/full/121/18/3002/DC1 Key words: Heat shock, Trypanosoma brucei, Stress granules, eIF2 alpha, P-bodies SummaryHeat shock causes a decrease in polysomes and the appearance of stress granules in trypanosomes independently of eIF2α phosphorylation at Thr169 Journal of Cell Science 3003 Heat shock stress granules and P-bodies in trypanosomes from a single promoter (Johnson et al., 1987; Kooter et al., 1987;Martinez-Calvillo et al., 2004;Martinez-Calvillo et al., 2003), and monocistronic mRNAs result from trans-splicing of a short, capped leader to the 5Ј end and linked 3Ј cleavage and polyadenylation of the upstream mRNA (Campbell et al., 1984;LeBowitz et al., 1993;Liang et al., 2003;Matthews et al., 1994;Schürch et al., 1994;Ullu et al., 1993). Consequently, the regulation of gene expression in trypanosomes is predominantly post-transcriptional (Clayton and Shapira, 2007). There is evidence for the presence of P-bodies: DHH1, XRNA and one Pumilio-family protein are located in cytoplasmic granules in normally growing cells (Caro et al., 2006;Cassola et al., 2007;Dallagiovanna et al., 2007;Dallagiovanna et al., 2005;Holetz et al., 2007), although any role in the regulation of gene expression has yet to be determined. In contrast to mRNA synthesis, the mechanisms of translation initiation and elongation appear to be typical for a eukaryote; all the factors identified in metazoa and yeast are present in the trypanosome genome (Ivens et al., 2005), although a functional analysis has only been performed on a small number (Dhalia et al., 2006;Dhalia et al., 2005) and very little is known about regulation of translation (Clayton and Shapira, 2007).Little is known about how the overall rate of gene expression is regulated, for example when trypanosomes stop growth and enter stationary phase. The only such phenomenon that has been investigated in any detail is the response to heat sh...

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In the mammalian host, the cell surface of Trypanosoma brucei is protected by a variant surface glycoprotein that is anchored in the plasma membrane through covalent attachment of the COOH terminus to a glycosylphosphatidylinositol. The trypanosome also contains a phospholipase C (GPI-PLC) that cleaves this anchor and could thus potentially enable the trypanosome to shed the surface coat of VSG. Indeed, release of the surface VSG can be observed within a few minutes on lysis of trypanosomes in vitro. To investigate whether the ability to cleave the membrane anchor of the VSG is an essential function of the enzyme in vivo, a GPI-PLC null mutant trypanosome has been generated by targeted gene deletion. The mutant trypanosomes are fully viable; they can go through an entire life cycle and maintain a persistent infection in mice. Thus the GPI-PLC is not an essential activity and is not necessary for antigenic variation. However, mice infected with the mutant trypanosomes have a reduced parasitemia and survive longer than those infected with control trypanosomes. This phenotype is partially alleviated when the null mutant is modified to express low levels of GPI-PLC.