The use of the Xenopus oocyte as a model system to analyze the expression and function of eukaryotic heat shock proteins

Biotechnology Advances 25 (2007) 385 – 395 www.elsevier.com/locate/biotechadv Research review paper The use of the Xenopus oocyte as a model system to analyze the expression and function of eukaryotic heat shock proteins John J. Heikkila a,⁎, Angelo Kaldis a , Genevieve Morrow b , Robert M. Tanguay b b a Department of Biology, University of Waterloo, Waterloo, ON, Canada N2L 3G1 Laboratoire de Génétique Cellulaire et Développementale, Dép. de Médecine CREFSIP, Pav. C.E.-Marchand, Université Laval, Québec, QC, Canada G1K 7P4 Received 9 February 2007; received in revised form 21 March 2007; accepted 21 March 2007 Available online 28 March 2007 Abstract The analysis of the expression and function of heat shock protein (hsp) genes, a class of molecular chaperones, has been greatly aided by studies carried out with Xenopus oocytes. The large size of the oocyte facilitates microinjection of DNA, mRNA or protein, permits manual dissection of nuclei, and allows certain assays to be performed with single oocytes. These and other characteristics were useful in identifying the cis- and trans-acting factors involved in hsp gene transcription as well as the role of chaperones and co-chaperones in the repression and activation of heat shock factor. Xenopus oocytes were used to examine heat shock protein (HSP) molecular chaperone function as well as their involvement in intracellular trafficking, maturation, and secretion of protein. Possible new areas of research with this system include the role of membranes in the heat shock response, involvement of HSPs in viral replication and maturation, and in vivo NMR spectroscopy of microinjected HSPs. © 2007 Elsevier Inc. All rights reserved. Keywords: Xenopus; Oocyte; mRNA; Microinjection; Heat shock proteins; Molecular chaperones; Gene expression Contents 1. 2. 3. 4. 5. 6. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Heat-inducible expression of hsp genes after microinjection into Xenopus oocytes Analysis of the mechanism of heat shock factor activation of hsp gene expression Analysis of the molecular chaperone function of HSPs in Xenopus oocytes . . . . Involvement of HSPs in translocation of protein and secretion . . . . . . . . . . 5.1. HSC70 recycling across the nuclear membrane . . . . . . . . . . . . . . . 5.2. Involvement of HSP70 and HSC70 on intracellular protein trafficking . . . 5.3. HSP90 involvement in nuclear export and MAP kinase pathway activation Possible future studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Membrane involvement of HSPs and the heat shock response . . . . . . . 6.2. Involvement of HSPs in viral replication and maturation . . . . . . . . . . 6.3. In vivo NMR spectroscopy of HSPs in microinjected Xenopus oocytes . . ⁎ Corresponding author. Tel.: +1 519 885 1211x33076; fax: +1 519 746 0614. E-mail address: heikkila@sciborg.uwaterloo.ca (J.J. Heikkila). 0734-9750/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.biotechadv.2007.03.003 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 386 387 388 389 390 390 390 391 391 391 392 392 386 J.J. Heikkila et al. / Biotechnology Advances 25 (2007) 385–395 7. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Introduction Our understanding of the flow of genetic information within a cell has been greatly aided by the ability to test the transcriptional efficiency of genes and the translational capacity of mRNAs after their microinjection into Xenopus oocytes. Furthermore, numerous studies have shown that protein, protein complexes, and antibodies and organelles (e.g. ribosomes, nuclei) can be microinjected into oocytes to study protein and/or cellular function (Heikkila, 1990; Kay and Peng, 1991; Gurdon, 2006; Liu, 2006). Even membrane fractions can be successfully transplanted from a variety of species into the membranes of Xenopus oocytes and still retain their original functionality (e.g. associated neurotransmitter receptors and voltage-operated channels) (Miledi et al., 2002, 2004). There are a number of characteristics associated with the Xenopus oocyte, an egg-forming cell in the ovary, which makes it an excellent experimental system. The large size of the oocyte (1–1.2 mm in diameter) facilitates microinjection of either DNA into the nucleus (germinal vesicle) or mRNA into the cytoplasm. In fact, the estimated internal volume of a mature oocyte is 0.54 ul, which is equivalent to approximately 200,000 somatic cells (Gurdon and Wakefield, 1986). Also, given the large size of the oocyte, it is relatively easy to manually dissect oocyte nuclei as well as to carry out various assays with single oocytes. The mature oocyte contains large amounts of the components required for gene expression including RNA polymerases, ribonucleotide triphosphates, transcription factors, histones, and ribosomes. For example, it has been estimated that a mature oocyte contains enough RNA polymerase to sustain an embryo to the 30,000-cell stage (Coleman, 1984a, b). Finally, the equipment required for Xenopus oocyte isolation, culture, and microinjection is relatively simple and inexpensive. Various protocols, outlining oocyte isolation, manipulation, fractionation, microinjection and other techniques, have been published (Table 1). In microinjection studies with Xenopus oocytes, the maximum microinjection volumes for cytoplasm and nucleus are typically 50 nl and 10 nl, respectively (Coleman, 1984a,b). The quantity of RNA microinjected into the cytoplasm usually ranges from 5 to 100 ng with lesser amounts required for polyadenylated, viral, and synthetic mRNA compared to total 393 393 393 RNA (Coleman, 1984b; Wormington, 1991; Gamarnik and Andino, 1996). The amount of DNA routinely microinjected into oocyte nuclei is 10 ng or less (Coleman, 1984a) while the amount of protein (recombinant protein, antibodies, cell extract, membrane preparations) injected into the cytoplasm can range up to several hundred nanograms (Schlatter et al., 2002; Miledi et al., 2002). Microinjection of purified or recombinant protein into the nucleus is usually at a lower range of approximately 1–20 ng or more (Wu et al., 1987; Bharadwaj et al., 1999). The laboratory of John Gurdon was the first to demonstrate that heterologous mRNAs, such as rabbit reticulocyte 9S mRNA, were capable of being translated in Xenopus oocytes (Gurdon et al., 1973). It was estimated that this RNA was translated 1000 times more efficiently in Xenopus oocytes than in a rabbit reticulocyte lysate in vitro translation system over an extended time period. Since that pioneering work Xenopus oocytes have been used to translate microinjected mRNA isolated from a wide range of organisms including amphibians, fish, birds, mammals, plants and viruses (Heikkila, 1990). In studies with microinjected synthetic mRNA, it was estimated that approximately 10 ng of protein per hour per oocyte was efficiently translated (Wormington, 1991). Another advantage of the Xenopus oocyte translation system is Table 1 Xenopus laevis oocyte methodology Technique Selected references Oocyte manipulation Kay and Peng, 1991; Liu, 2006 Coleman, 1984b; Wormington, 1991 Coleman, 1984a; Gurdon and Wakefield, 1986 Geib et al., 2001; Tokmakov et al., 2006 Gurdon (2006) Abdulle et al., 2002; Kaldis et al., 2004 Evans and Kay, 1991; Paine et al., 1992 Goldin, 1991; Cucu et al., 2004 Miledi et al., 2002, 2004 Selenko et al., 2006; Sakai et al., 2006 mRNA microinjection DNA microinjection Coupled transcription-translation Nuclear transplantation Protein refolding Isolation of nuclei Electrophysiological analysis Membrane transplantation In vivo NMR spectroscopy J.J. Heikkila et al. / Biotechnology Advances 25 (2007) 385–395 its ability to post-translationally modify newly synthesized proteins (e.g. glycosylation, phosphorylation and cleavage of protein precursors). For example, microinjection of a synthetic mRNA encoding the human growth factor receptor resulted in correct synthesis, processing, glycosylation and transport to the cell surface of the Xenopus oocyte (Seghal et al., 1988). The ability to post-translationally modify protein is a property not shared by most in vitro translation systems. A drawback of this protocol of injecting specific mRNAs is the time and effort required to produce synthetic mRNA. However, a new technique circumvents this problem by the co-injection into the cytoplasm of Xenopus oocytes a cDNA cloned into a plasmid vector downstream of a T7 RNA polymerase promoter and T7 RNA polymerase (Geib et al., 2001; Tokmakov et al., 2006). This method results in the efficient transcription of the injected cDNA by T7 RNA polymerase and translation of the resultant mRNA by the host protein synthetic machinery. As mentioned previously, the Xenopus oocyte has been used to examine the expression of injected DNA. Coleman (1975) first reported that the Xenopus oocyte was capable of transcribing microinjected polynucleotides. This was followed by the finding that SV40 DNA was transcribed efficiently when injected into the germinal vesicle of the oocyte and the resultant mRNA was correctly translated into viral protein (DeRobertis and Mertz, 1977). Over the last 30 years, the Xenopus oocyte has been used to transcribe numerous microinjected genes from diverse organisms (Heikkila, 1990; Liu, 2006). Caution should be drawn to the fact that genes requiring specific transcription factors not normally found in the Xenopus oocyte may not be transcribed correctly. However, this drawback can be overcome by the co-injection of the trans-acting factor(s) or the mRNA that encodes it (Heikkila, 1990). While the Xenopus oocyte has been used to study the expression and function of a variety of different genes from a wide range of organisms, the present review will illustrate the importance of the Xenopus oocyte as a model system by examining its use in the analysis of heat shock protein (hsp) gene expression and function. The synthesis of heat shock proteins (HSPs) is elevated in cells in response to environmental stresses including elevated temperature and exposure to heavy metals (Feige et al., 1996; Morimoto, 1998). A subset of HSPs is expressed normally within the cell and function as molecular chaperones. During cellular stress, HSPs bind to and inhibit irreversible aggregation or misfolding of denatured or damaged proteins as well as facilitating their refolding once favorable environmental conditions 387 have returned. Numerous studies have shown that protein aggregation is detrimental to a living cell and that it has been implicated in a number of human diseases including Alzheimer's, muscle myopathy, and multiple sclerosis (Feige et al., 1996; Quinlan and Van Den Ijssel, 1999; Bova et al., 1999). There are 3 major families of HSPs; the high molecular weight HSPs (e.g. HSP90), HSP70, and the small HSPs (sHSPs) (Feige et al., 1996; Morimoto, 1998). The HSP70 family consists of cytoplasmic HSP70 and HSC70, mitochondrial HSP70, and BiP, the resident endoplasmic reticulum (ER) immunoglobulin-binding protein (also known as glucose-regulated protein 78). Stress-induced hsp gene expression involves interaction of the heat shock element enhancer (HSE; generally present in the promoter region of hsp genes with the exception of hsc70 in some species) with heat shock transcription factor (HSF; Voellmy, 2004). HSF preexists as an inactive monomer that undergoes heat shock-induced trimer formation and binding to the HSE thus facilitating transcription. While eukaryotic cells contain multiple HSF family members, HSF1 appears to be the primary transcription factor that mediates stress-induced expression of hsp genes including, hsp90, hsp70, and shsps families (Voellmy, 2004). 2. Heat-inducible expression of hsp genes after microinjection into Xenopus oocytes The Xenopus oocyte model system has been of great value in unraveling the mechanisms associated with hsp gene expression. The work of Voellmy and Rungger (1982) was the first study to show that a Drosophila hsp gene, hsp70, when injected into Xenopus oocyte nuclei was capable of efficiently transcribing the gene in a heat-inducible manner. Furthermore, the transcription of hsp70 was sensitive to α-amanatin, indicating that it was transcribed by RNA polymerase II. This result was confirmed by Bienz and Pelham (1982) who performed deletion mutant analysis of the 5′ upstream region, demonstrating that the DNA sequence required for heatinducible transcription occurred between 10 and 66 bases upstream from the transcriptional start site and that it was a motif common to other hsp gene promoters. This sequence element was eventually termed the HSE. Pelham and Bienz (1982) further reported that placement of a synthetic double-stranded oligonucleotide containing the HSE upstream of the TATA box of the herpes virus thymidine kinase gene resulted in heatinducible expression after microinjection into Xenopus oocytes. Similar Xenopus oocyte microinjection studies 388 J.J. Heikkila et al. / Biotechnology Advances 25 (2007) 385–395 showing heat inducible expression were carried out with Xenopus hsp30 and hsp70 genes as well as Drosophila hsp23, and human hsp70 genes (Bienz, 1984; Mestril et al., 1985; Voellmy et al., 1985; Dreano et al., 1986). All of the aforementioned studies used an elevation in incubation temperature to activate hsp gene expression. Ananthan et al. (1986) provided the first indication that the presence of elevated levels of denatured protein could trigger the heat shock response. In this study, a Drosophila hsp70 gene promoter fused to a β-galactosidase gene was microinjected into Xenopus oocyte nuclei. While co-injection of native protein such as bovine serum albumin or bovine β-lactoglobulin had no effect on the expression of the injected gene, injection of heat-denatured protein induced the transcription of the Drosophila hsp70/β-galactosidase gene construct. This pivotal observation, which provided insight on the mechanism of induction of hsp gene transcription, was extended further by Mifflin and Cohen (1994a). This latter study determined that the ability of denatured protein to activate hsp gene expression in Xenopus oocytes correlated with protein aggregation and longevity in the cell as well as microinjection directly into the nucleus. 3. Analysis of the mechanism of heat shock factor activation of hsp gene expression The transcription factor responsible for heat shockinduced transcription of hsp genes, namely HSF, was first purified from Drosophila cultured cells (Wu et al., 1987). This study assayed the functional properties of Drosophila HSF by microinjection into Xenopus oocytes. They found that transcription of a microinjected Drosophila hsp70 gene was stimulated 30-fold by a subsequent injection of purified Drosophila HSF into Xenopus oocytes maintained at control temperatures. Wu et al. (1987) also determined that the start of the transcripts were identical with hsp70 transcripts made in vivo in heat shocked Drosophila cells. The Xenopus oocyte system was also used to examine the properties of human HSF1 (Baler et al., 1993). In this study, synthetic HSF1 mRNA was microinjected into Xenopus oocyte cytoplasm, allowed to translate for 1– 2 days and then assessed for HSE DNA binding activity. Human HSF1 derived from heat shocked but not control oocytes displayed high affinity HSE DNA binding activity. Subsequent studies determined that heat shock induced the formation of homotrimeric complexes that were essential for its HSE DNA binding activity in Xenopus oocytes (Zuo et al., 1994, 1995). In these latter studies, the authors carried out a mutagenic analysis of human HSF1 to investigate the protein domains required for trimerization and DNA binding. The Xenopus oocyte system has also been used to examine the properties of Xenopus HSF. Earlier studies documented the presence of heat-activatable HSF–HSE binding activity in eggs and embryos of Xenopus laevis (Ovsenek and Heikkila, 1990). An examination of endogenous HSF1 during X. laevis oogenesis was carried out by Gordon et al. (1997) who found that the properties of oocyte HSF1 were distinct from those found in eggs or embryos. For example, heat-induced DNA-binding activity of HSF1 in oocytes, which was correlated with the heat-induced expression of a microinjected hsp70 promoter construct, declined rapidly during recovery at the control temperature. However, prolonged stress treatments maintained elevated HSF–HSE binding activity, which was distinct from the transient HSE– HSF binding activity found in eggs and embryos under similar conditions (Ovsenek and Heikkila, 1990). There have been conflicting reports with respect to the intracellular localization of HSF1 in eukaryotic cells. Some reports have suggested that HSF1 was cytoplasmic and migrated into the nucleus upon heat shock (Martinez-Balbas et al., 1995). Others reported that HSF1 was localized primarily in the nucleus prior to stress (Westwood et al., 1991). This area of investigation was analyzed in Xenopus oocytes because of their extremely large size and the fact that one can physically dissect and isolate nuclei with just the aid of a dissecting microscope and appropriate tools. Mercier et al. (1997) used this approach to show that HSF1 binding activity was not detectable in isolated nuclei but that it could be induced in vitro by elevated temperature. Furthermore, enucleated oocytes did not show any heat-inducible HSF1 binding activity. This study suggests that in Xenopus oocytes, HSF1 was located within the nucleus prior to HSF activation. In a recent review examining the mechanisms that are involved in controlling stress-induced HSF1 activity in metazoan cells, it was suggested that the regulation of HSF1 activity occurs at multiple levels (Voellmy, 2004), including oligomerization of HSF1, posttranslational modifications, transcriptional competence, nuclear/subnuclear localization and interaction with regulatory cofactors or other transcription factors. Studies using the Xenopus oocyte system have shown that molecular chaperones and co-chaperones can regulate the heat shock response. For example, Mifflin and Cohen (1994b) reported that injections of HSC70 protein into Xenopus oocytes lowered the stress response to both a thermal shock and co-injected protein inducers. Also Ali et al. (1998) demonstrated that the molecular chaperone, J.J. Heikkila et al. / Biotechnology Advances 25 (2007) 385–395 HSP90, functions in the regulation of HSF1 in Xenopus oocytes. Co-immunoprecipitation experiments with extract obtained from manually dissected nuclei revealed the presence of HSP90 associated with HSF1. Furthermore, microinjection of HSP90 antibodies directly into oocyte nuclei inhibited heat-induced transcriptional activity of HSF1. These studies suggested that HSP90 interacts with the inactive monomeric and active trimeric forms of HSF1 and could be involved in regulating the interconversion between these forms as well as repressing HSF1 activity. Subsequent studies suggested that additional factors were negative modulators of HSF1 transcriptional activity in Xenopus oocytes including the co-chaperones, p23, FKBP52 plus glycogen synthase kinase 3β and protein phosphatase 5 (Bharadwaj et al., 1999; Xavier et al., 2000; Conde et al., 2005). These aforementioned studies illustrate the benefits of using the Xenopus oocyte system to understand the stress activated heat shock response. In the future, this system will be of value in examining in vivo the numerous unresolved questions regarding HSF1 activation such as the role of specific kinases in the pathway leading to HSF1 phosphorylation, the role of additional chaperone and co-chaperone molecules, the function of HSF1 sumoylation, and interaction with other transcription factors. 389 researchers have traditionally employed in vitro assays using either rabbit reticulocyte or wheat germ lysates as a source of molecular chaperones (Lee and Vierling, 1998; Abdulle et al., 2002; Morrow et al., 2006). Recently, an assay to assess the ability of a molecular chaperone to maintain denatured client protein, firefly luciferase (LUC), in a folding competent state was developed using microinjected Xenopus oocytes (Abdulle et al., 2002; Fernando et al., 2002). In this assay, microinjection of heat denatured LUC into oocytes regained very little enzyme activity over time. However, heat denaturation of LUC in the presence of Xenopus sHSP, HSP30, resulted in 100% enzyme reactivation following oocyte injection. Xenopus oocytes contain a variety of molecular chaperones involved in protein folding including HSP70 (Bienz, 1984; Uzawa et al., 1995; Abdulle et al., 2002). This in vivo refolding assay required less molecular chaperone and substrate than rabbit reticulocyte or wheat germ lysate in vitro refolding assays (Abdulle et al., 2002). In addition this assay was used successfully with sHSPs from other organisms including the American bullfrog, Rana catesbeiana (Kaldis et al., 2004) and the fruit fly, Drosophila melanogaster. An example of the results obtained with this assay using Drosophila HSP22 and HSP23 is shown in Fig. 1. Heat treatment of LUC alone 4. Analysis of the molecular chaperone function of HSPs in Xenopus oocytes HSPs have become recognized as a critical component of the intracellular environment. These molecular chaperones have the ability to aid in the folding and translocation of cellular proteins (Feige et al., 1996; Morimoto, 1998). An important function of HSPs is their ability to bind and stabilize proteins that are partially denatured in response to environmental stress and maintain these client proteins in a state that inhibits aggregation and permits them to regain proper structure and function once favorable cellular conditions have been reestablished. A number of studies have shown that molecular chaperones such as members of the HSP70 family and HSP60 are involved in the refolding of unfolded client protein back into an active form (Feige et al., 1996). Various protocols have been developed to assess the ability of purified or recombinant HSPs to inhibit heat- or chemical-induced target protein aggregation in vitro using light scattering (Lee and Vierling, 1998; Fernando and Heikkila, 2000; Morrow et al., 2006). To determine whether an HSP maintains its denatured client protein in a folding competent state, Fig. 1. Ability of LUC heat denatured with Drosophila sHSPs to be reactivated in vivo after microinjection into Xenopus oocytes. Drosophila recombinant HSP22 and HSP23 were purified as previously described (Morrow et al., 2006). Luciferase (0.2 μM) was incubated at 42 °C for 8 min either alone (solid diamond) or with 1 μM bovine serum albumin, Drosophila HSP22 (solid triangle), or Drosophila HSP23 (open triangle). Samples containing 1.38 fmol of LUC in 27.6 nl were microinjected into Xenopus oocytes and LUC activity was monitored as described in Kaldis et al. (2004). Data are expressed as a percentage of the activity of native LUC after microinjection into Xenopus oocytes and presented as the mean ± S.E. of 4–5 trials. 390 J.J. Heikkila et al. / Biotechnology Advances 25 (2007) 385–395 or in the presence of bovine serum albumin regained little enzyme activity after microinjection into Xenopus oocytes. However, performing the heat denaturation of LUC in the presence of HSP22 or HSP23 resulted in complete recovery of LUC activity in oocytes. It is likely that this assay will have applications for a variety of molecular chaperones from different organisms. Also coupling this assay with deletion or sitespecific mutagenesis of the HSP will allow the determination of protein domains essential for maintenance of the client protein in a folding competent state (Fernando et al., 2002). 5. Involvement of HSPs in translocation of protein and secretion 5.1. HSC70 recycling across the nuclear membrane As mentioned previously, the Xenopus oocyte model system was used to demonstrate the presence of HSF and HSP90 in the nucleus. Additionally, the oocyte microinjection system was employed to study the intracellular localization of other HSPs including HSC70. Mandell and Feldherr (1990) showed that HSC70 was capable of moving back and forth across the nuclear membrane of Xenopus oocytes. For example, microinjection of radioactively-labeled rat HSC70 into the oocyte cytoplasm led to an enrichment in nuclei after 6 h by 24-fold compared to bovine serum albumin controls. Also, HSC70 microinjected directly into oocyte nuclei resulted in a relocation into the cytoplasm at a greater rate than bovine serum albumin. These observations were verified in transfected COS7 cells showing that human HSC70 possessed both a nuclear targeting sequence as well as a nuclear export signal (Tsukahara and Maru, 2004). 5.2. Involvement of HSP70 and HSC70 on intracellular protein trafficking HSC70 and HSP70 are also associated with the intracellular localization of client proteins. A recent study has shown that HSP70 and HSC70 have different effects on the functional expression and intracellular trafficking of murine epithelial sodium channels in Xenopus oocytes (Goldfarb et al., 2006). In this study, mRNA encoding the three subunits of the epithelial sodium channel were microinjected into Xenopus oocytes and the formation of the channels was assessed by voltage clamp analysis. The coinjection of mRNA encoding HSC70 inhibited the formation of the epithelial sodium channels, whereas co-injection of HSP70 mRNA enhanced their production. While the underlying mechanism(s) for these results is not known at present, these data contradict earlier suggestions that HSP70 and HSC70 are functionally interchangeable. The synthesis and maturation of integral membrane and secretory proteins involve a number of ordered steps including translation, translocation into the ER, cotranslational modification, conformational maturation, multimerization and screening for quality control prior to transport to the Golgi apparatus. The resident ER member of the HSP70 family, BiP, is thought to have an important function(s) during the biosynthesis and ER residency of secretory and transmembrane proteins. Beggah et al. (1996) examined the association of BiP with the two subunits of Na,K-ATPase in Xenopus oocytes. The α-subunit is the ATP-hydrolyzing catalytic subunit embedded within the membrane or present in the cytoplasm while the β-subunit is a type II glycoprotein that traverses the membrane and has a large ectodomain that is modified by disulfide bridges and carbohydrate additions. Results from a series of RNA microinjection and co-immunoprecipitation experiments demonstrated that BiP associated with βsubunits until assembly with α-subunits occurred. The use of various wild type and mutant β-subunits revealed that their temporal association with BiP corresponded to the stability of the various β-subunit proteins. This study supported the role of BiP as a molecular chaperone and that it was part of the ER process of maturation of a multi-membrane-spanning protein. Cortical granule exocytosis is an early postfertilization event that leads to a sustained block of polyspermy in Xenopus eggs (Bement and Capco, 1989; Lindsay and Hedrick, 1989; Chang et al., 1999). In this process, cortical granules undergo exocytosis such that they release proteases that cleave the proteins linking the vitelline envelope to the cell membrane. Mucopolysaccharides released by the cortical granules create an osmotic gradient causing water to enter and swell the space between the cell membrane and fertilization envelope. Cysteine string proteins appear to be involved in the fusion of these granules with the cell membrane. Recent studies suggest that the normal pathway for cortical granule exocytosis involves an interaction between granule-associated cysteine string proteins and HSC70 in Xenopus oocytes (Smith et al., 2005). They observed that the J-domain of the cysteine string protein binds to HSC70 and that overexpression of the Jdomain by RNA microinjection inhibited cortical granule secretion. However, the inhibition was eliminated by injecting mutant J-domain which did not J.J. Heikkila et al. / Biotechnology Advances 25 (2007) 385–395 interact with HSC70. These experiments revealed in Xenopus oocyte that interaction between HSC70 and cysteine string protein was important for cortical granule exocytosis. 5.3. HSP90 involvement in nuclear export and MAP kinase pathway activation As mentioned previously, the Xenopus oocyte system was used to demonstrate that HSP90 was involved the process of interconversion of HSF molecules from inactive monomers to active trimers. It has been suggested that HSP90 may also be involved in facilitating the nuclear export of 60 S ribosomal subunits in Xenopus oocytes (Schlatter et al., 2002). Ribosomal subunits, which are assembled in the nucleus, are exported stoichiometrically into the cytoplasm in a unidirectional, saturable and energy-dependent process. Microinjection of geldanamycin or HSP90 antibody (both of which inhibit HSP90 function) into the Xenopus oocyte cytoplasm markedly reduced the rate of export of microinjected rat liver 60 S ribosomal subunits from the nucleus to the cytoplasm. This study illustrates the involvement of HSP90 in 60S ribosomal subunit translocation from the nucleus to the cytoplasm in Xenopus oocytes. In vertebrate oocytes, the proto-oncogene, Mos, activates the MAP kinase cascade during meiosis. Mos-mediated control of MAP kinase appears to be critical for the correct ordering and timing of events during early development such as suppression of replication in meiosis and prevention of parthenogenetic activation. It was shown in Xenopus oocytes by means of a series of glutathione-S-transferase (GST) pull-down assays that HSP90 was required for Mos and MAP kinase activation (Fisher et al., 2000). For example, GST-Mos mRNA was microinjected into Xenopus oocytes and translated. Pull-down assays of oocyte homogenates determined that Mos interacted with HSP90 and HSP70. Also treatment of oocytes with geldanamycin, an inhibitor of HSP90 function, blocked the association of HSP90 with Mos as well as abolishing its kinase activity. These and other experiments revealed that HSP90 was required for activation and phosphorylation of Mos and full activation of the MAP kinase cascade. 6. Possible future studies As outlined in this review, there are numerous unresolved questions with respect to hsp gene expression and the function of HSPs within the cell. The 391 following section examines the potential use of the Xenopus oocytes in three selected areas of hsp gene research. 6.1. Membrane involvement of HSPs and the heat shock response Recent studies have shown that stress-induced membrane perturbations activate signal(s) leading to the transcription of hsp genes (Torok et al., 2003; Balogh et al., 2005). It was shown that hsp gene expression can be initiated at lower temperatures in cells with more fluid membranes. Furthermore, the association of membranes with HSPs, such as sHSPs, may stabilize membranes leading to a down-regulation of hsp gene expression. It was suggested that even subtle alterations or defects of the lipid phase of membranes that occur with ageing or disease states could influence membrane-initiated signaling processes, leading to a dysregulated stress response. The analysis of the contribution of membrane fluidity or composition to the heat shock response could be examined in a novel way with Xenopus oocytes. A remarkable property of Xenopus oocytes is its ability to receive transplanted membrane fractions. For example, Marsal et al. (1995) injected cell membranes from the electric organ of the pacific electric ray, Torpedo californica, into Xenopus oocytes. The injected membrane vesicles from Torpedo fused with the oocyte membrane and caused the appearance of functional Torpedo acetylcholine receptors and chloride channels. In a more recent study, temporal neocortex membranes obtained postoperatively from a patient with intractable epilepsy were microinjected into oocytes and within a few hours acquired functional neurotransmitter receptors including those binding γ-aminobutyric acid, α-amino3-hybroxy-5-methyl-4-isoxazolepropionic acid, kainate, and glycine (Miledi et al., 2002). These receptors were also formed if mRNA extracted from the temporal neocortex of the same patient was injected into Xenopus oocytes. Moreover, there is the potential to investigate postmortem membranes that have been kept frozen for many years. Miledi et al. (2004) has shown that membranes isolated from frozen brain tissue of Alzheimer's patients could be microtransplanted into Xenopus oocytes and that the associated neurotransmitter receptors and voltage-operated channels including γ-aminobutyric acid (GABA) receptors were still functional. Microinjection of cell membranes into Xenopus oocytes complements mRNA or DNA microinjection protocols because the foreign membranes fuse with the oocyte's own membrane while 392 J.J. Heikkila et al. / Biotechnology Advances 25 (2007) 385–395 still maintaining the original receptors and associated molecules embedded in their natural lipid environment. This newer technology makes the Xenopus oocyte a useful model system to study the impact of transplanted membrane on the activation of HSF1 and transcription of a microinjected hsp promoter/reporter gene construct. This membrane material could be obtained from cultured cells experimentally modified prior to injection or obtained from tissues of patients with various disease states. 6.2. Involvement of HSPs in viral replication and maturation Numerous studies have demonstrated that HSPs are associated with the replication and maturation of viruses (Mayer, 2005). Most viruses require molecular chaperones including HSPs during their life cycle. For the successful viral production in a host cell, a virus needs to properly fold its own protein as well as interfere with a number of cellular processes including signal transduction, cell cycle regulation, and induction of apoptosis. HSPs are intimately involved in the control of these cellular processes. Indeed some viruses actively recruit cellular HSPs during their life cycle including HSP70, HSC70, and HSP90. Xenopus oocytes have also been used to study the process of viral replication and maturation. For example, Zhou et al. (1992) obtained the first insight into the assembly of hepatitis B virus capsid particles following microinjection of capped mRNA into Xenopus oocytes. Subsequently, Gamarnik and Andino (1996) demonstrated that microinjection of a poliovirus RNA into Xenopus oocytes initiated a complete cycle of viral replication. This included translation of the viral RNA to produce structural proteins for capsid formation and nonstructural viral proteins for genome replication, as well as virus assembly, yielding a high level of infectious viral particles. However, two human protein factors, one involved in translational initiation while the other was associated with RNA synthesis, needed to be co-injected for poliovirus replication to occur. A similar requirement of human cellular extract was noted for the production of high levels of human rhinovirus 14 infection particles following injection of its RNA (Gamarnik et al., 2000; Gamarnik and Andino, 2006). In contrast, microinjection of the RNA encoding mengovirus or Theiler's murine encephalomyelitis virus into Xenopus oocytes resulted in the production of infectious virus particles without the need for additional co-factors (Gamarnik et al., 2000; Franco et al., 2005). As indicated in the aforementioned studies, the Xenopus oocyte is a useful tool to study viral biology. These studies also suggest that the Xenopus oocyte may be an ideal system to study the involvement of HSPs in viral replication and maturation. 6.3. In vivo NMR spectroscopy of HSPs in microinjected Xenopus oocytes The analysis of the structure of a purified protein is typically confined to in vitro experimental set ups (e.g. X-ray crystallography or cryo-electron microscopy) which give useful but limited information. It should be noted that in vivo proteins are not trapped in one particular conformation but change their structure dynamically within a cell as a result of chemical modification or formation of complexes with other proteins, membrane phospholipids or nucleic acids. Furthermore, structural changes of proteins in vivo may be closely related to their function as well as to certain cellular events. In-cell or in vivo NMR spectroscopy has been used to examine the structural and functional characteristics of cellular protein, protein dynamics and protein–protein interactions in bacterial cells (Bryant et al., 2005; Burz et al., 2006). Recently, in vivo NMR spectroscopy was successfully applied to the examination of 15N-labeled proteins microinjected into Xenopus oocytes (Selenko et al., 2006; Sakai et al., 2006). For example, NMR spectroscopy was able to follow the protein–protein interaction of calmodulin and ubiquitin as well as the maturation of ubquitin precursor derivatives in living oocytes. It is possible that this technique could be applied to the study of the functional interaction of 15N-labeled HSPs with client proteins or protein complexes. Additionally, it may be possible to Table 2 Function of different eukaryotic HSPs determined in Xenopus oocytes HSP Function and reference HSP90 Regulation of HSF activity (Ali et al., 1998) Nuclear export of rat liver 60 S ribosomal subunits (Schlatter et al., 2002) Mos and MAP kinase activation (Fisher et al., 2000) Cortical granule exocytosis (Smith et al., 2005) Intracellular protein trafficking (Goldfarb et al., 2006) HSC70 recycling across nuclear membrane (Mandell and Feldherr, 1990) HSC70 regulation of stress response (Mifflin and Cohen, 1994b) Protein maturation in endoplasmic reticulum (Beggah et al., 1996) Maintenance of client protein in folding competent state (Abdulle et al., 2002; Fernando et al., 2002; Kaldis et al., 2004) HSP/HSC70 BiP sHSP J.J. Heikkila et al. / Biotechnology Advances 25 (2007) 385–395 follow the process of microinjected HSF1 trimerization as well as the interaction of HSF1 with co-chaperones or other regulatory molecules in oocytes or manually dissected oocyte nuclei. 7. Conclusions The ability to microinject hsp gene promoter/ reporter genes, hsp mRNAs or HSPs into X. laevis oocytes has greatly contributed to our knowledge of hsp gene expression and function (Table 2). It is apparent that the large size of the Xenopus oocyte simplifies the techniques of microinjection, manual dissection of individual nuclei, and assays performed on individual oocytes (e.g. HSF–HSE binding activity, patch clamp analysis of membrane transporters, or single cell assays). Undoubtedly, the Xenopus oocyte model system will continue to be a useful addition to the growing arsenal of molecular and cell biology techniques. Acknowledgements This research was supported by the Natural Science and Engineering Research Council (NSERC) grants to J. J.H. who is also the recipient of a Canada Research Chair in Stress Protein Gene Research. This work was also supported by a grant from the Canadian Institutes of Health Research (CIHR) to RMT and CIHR and FRSQFCAR studentships to GM. References Abdulle R, Mohindra A, Fernando P, Heikkila JJ. Xenopus small heat shock protein hsp30C and hsp30D, maintain heat and chemically denatured luciferase in a folding-competent state. Cell Stress Chaperones 2002;7:6–16. Ali A, Bharadwaj S, O'Carrol R, Ovsenek N. HSP90 interacts with and regulates the activity of heat shock factor 1 in Xenopus oocytes. Mol Cell Biol 1998;18:4949–60. Ananthan J, Goldberg AL, Voellmy R. Abnormal proteins serve as eukaryotic stress signals and trigger the activation of heat shock genes. Science 1986;232:522–4. Baler R, Dahl G, Voellmy R. Activation of human heat shock genes is accompanied by oligomerization, modification, and rapid translocation of heat shock transcription factor HSF1. Mol Cell Biol 1993;13:2486–96. Balogh G, Horvath I, Nagy E, Zsofia E, Hoyk Z, Benko S, et al. The hyperfluidization of mammalian cell membranes acts as a signal to initiate the heat shock protein response. FEBS J 2005;272: 6077–86. Beggah A, Mathews P, Beguin P, Geering K. Degradation and endoplasmic reticulum retention of unassembled α- and β-subunits of Na,K-ATPase correlate with interaction of BiP. J Biol Chem 1996;271:20895–902. 393 Bement WM, Capco DG. Activators of protein kinase C trigger cortical granule exocytosis, cortical contraction, and cleavage furrow formation in Xenopus laevis oocytes and eggs. J Cell Biol 1989;108:885–92. Bharadwaj S, Ali A, Ovsenek N. Multiple components of the HSP90 chaperone complex function in regulation of heat shock factor 1 in vivo. Mol Cell Biol 1999;19:8033–41. Bienz M. Xenopus hsp70 genes are constitutively expressed in injected oocytes. EMBO J 1984;3:2477–83. Bienz M, Pelham HRB. Expression of a Drosophila heat shock protein in Xenopus oocytes: conserved and divergent regulatory signals. EMBO J 1982;1:1582–8. Bova MP, Yaron O, Huang QL, Ding LL, Haley DA, Stewart PI, et al. Mutation R120G in alphaB-crystallin, which is linked to a desminrelated myopathy, results in an irregular structure and defective chaperone-like function. Proc Natl Acad Sci U S A 1999;96: 6137–42. Bryant JE, Lecomte JT, Lee AL, Young GB, Pielak GJ. Protein dynamics in living cells. Biochemistry 2005;44:9275–9. Burz DS, Dutta K, Cowburn D, Shekhtman A. Mapping structural interactions using in-cell NMR spectroscopy. Nat Methods 2006;3:91–3. Chang P, Perez-Mongiovi D, Houliston E. Organization of Xenopus oocyte and egg cortices. Microsc Res Tech 1999;44:415–29. Coleman A. Transcription of DNAs of known sequence after injection into the eggs and oocytes of Xenopus laevis. Eur J Biochem 1975;113:339–48. Coleman A. Expression of exogenous DNA in Xenopus oocytes. In: Hames BD, Higgins SJ, editors. Transcription and translation: a practical approach. Washington, D.C.: IRL Press; 1984a. p. 49–69. Coleman A. Translation of eukaryotic messenger RNA in Xenopus oocytes. In: Hames BD, Higgins SJ, editors. Transcription and translation: a practical approach. Washington, D.C.: IRL Press; 1984b. p. 271–302. Conde R, Xavier J, McLoughlin C, Chinkers M, Ovsenek N. Protein phosphatase 5 is a negative modulator of heat shock factor 1. J Biol Chem 2005;280:28989–96. Cucu D, Simaels J, Jans D, Van Driessche W. The transoocyte voltage clamp: a non-invasive technique for electrophysiological experiments with Xenopus laevis oocytes. Pflugers Arch-Eur J Physiol 2004;447:934–42. DeRobertis EM, Mertz JE. Coupled transcription and translation of DNA injected into Xenopus oocytes. Cell 1977;12:175–82. Dreano M, Brochot J, Myers A, Cheng-Meyer C, Rungger D, Voellmy R, et al. High-level, heat-regulated synthesis of proteins in eukaryotic cells. Gene 1986;49:1–8. Evans JP, Kay BK. Biochemical fractionation of oocytes. Xenopus laevis: practical uses in cell and molecular biology. Methods in cell biologySan Diego: Academic Press Inc.; 1991. p. 133–48. Feige U, Morimoto RI, Yahara I, Polla BS. Stress-inducible cellular responses. Basel, Switzerland: Birkhauser Verlag; 1996. Fernando P, Heikkila JJ. Functional characterization of Xenopus small heat shock protein, hsp30C: the carboxyl end is required for stability and chaperone activity. Cell Stress Chaperones 2000;5: 148–59. Fernando P, Abdulle R, Mohindra A, Guillemette JG, Heikkila JJ. Mutation or deletion of the C-terminal tail affects the function and structure of Xenopus laevis small heat shock protein, hsp30. Comp Biochem Physiol 2002;133:95–103. Fisher DL, Mandart E, Doree M. Hsp90 is required for c-Mos activation and biphasic MAP kinase activation in Xenopus oocytes. EMBO J 2000;19:1516–24. 394 J.J. Heikkila et al. / Biotechnology Advances 25 (2007) 385–395 Franco D, Stevens I, Steurbaut S, Goris J, Rombaut B. Replication of Theiler's murine encephalomyelitis virus in Xenopus laevis oocytes. Virus Res 2005;107:35–8. Gamarnik AV, Andino R. Replication of poliovirus in Xenopus oocytes requires two human factors. EMBO J 1996;15:5988–98. Gamarnik AV, Andino R. Exploring RNA virus replication in Xenopus oocytes. Methods Mol Biol 2006;322:367–78. Gamarnik AV, Boddeker N, Andino R. Translation and replication of human Rhinovirus type 14 and Mengovirus in Xenopus oocytes. J Virol 2000;74:11983–7. Geib S, Sandoz G, Carlier E, Cornet V, Cheynet-Sauvion V, De Waard M. A novel Xenopus oocyte expression system based on cytoplasmic coinjection of T7-driven plasmids and purified T7RNA polymerase. Recept. Channels 2001;7:331–43. Goldfarb SB, Kashlan OB, Watkins JN, Suaud L, Yan W, Kleyman TR, et al. Differential effects of Hsc70 and Hsp70 in intracellular trafficking and functional expression of epithelial sodium channels. Proc Natl Acad Sci U S A 2006;103:5817–22. Goldin AL. Expression of ion channels by injection of mRNA into Xenopus oocytes. In: Kay BK, Peng HB, editors. Xenopus laevis: practical uses in cell and molecular biology. Methods in cell biologySan Diego: Academic Press Inc.; 1991. p. 487–509. Gordon S, Bharadwaj S, Hnatov A, Ali A, Ovsenek N. Distinct stressinducible and developmentally regulated heat shock transcription factors in Xenopus oocytes. Dev Biol 1997;181:47–63. Gurdon JB. Nuclear transplantation in Xenopus. Methods Mol Biol 2006;325:1–9. Gurdon JB, Wakefield L. Microinjection of amphibian oocytes and eggs for the analysis of transcription. In: Cellis JE, editor. Microinjection and organelle transplantation techniques. London: Academic Press Inc.; 1986. p. 269–99. Gurdon JB, Lingrel J, Marbaix G. Message stability in injected frog oocytes: long life of mammalian α and β globin messages. J Mol Biol 1973;80:539–51. Heikkila JJ. Expression of cloned genes and translation of messenger RNA in microinjected Xenopus oocytes. Int J Biochem 1990;22: 1223–8. Kaldis A, Atkinson BG, Heikkila JJ. Molecular chaperone function of the Rana catesbeiana small heat shock protein, Hsp30. Comp Biochem Physiol 2004;139:175–82. Kay BK, Peng HB. Xenopus laevis: practical uses in cell and molecular biology. Methods in cell biology, vol. 36. San Diego: Academic Press Inc.; 1991. Lee GJ, Vierling E. Expression, purification and molecular chaperone activity of plant recombinant small heat shock proteins. Methods Enzymol 1998;290:350–65. Lindsay LL, Hedrick JL. Proteases releasd from Xenopus laevis eggs at activation and their role in envelope conversion. Dev Biol 1989;135:202–11. Liu XJ. Xenopus protocols: cell biology and signal transduction. Methods in molecular biology, vol. 322. Totowa, NJ: Humana Press; 2006. Mandell RB, Feldherr CM. Identification of two HSP70-related Xenopus oocyte proteins that are capable of recycling across the nuclear envelope. J Cell Biol 1990;111:1775–83. Marsal J, Tigyi G, Miledi R. Incorporation of acetylcholine receptors and Cl− channels in Xenopus oocytes injected with Torpedo electroplaque membranes. Proc Natl Acad Sci U S A 1995;92: 5224–8. Martinez-Balbas MA, Dey A, Rabindran SK, Ozato K, Wu C. Displacement of sequence-specific transcription factors from chromatin. Cell 1995;83:29–38. Mayer MP. Recruitment of Hsp70 chaperones: a crucial part of viral survival strategies. Rev Physiol Biochem Pharmacol 2005;153: 1–46. Mercier PA, Foksa J, Ovsenek N, Westwwod JT. Xenopus heat shock factor 1 is a nuclear protein before heat stress. J Biol Chem 1997;272:14147–51. Mestril R, Rungger D, Schiller P, Voellmy R. Identification of a sequence element in the promoter of the Drosophila melanogaster hsp23 gene that is required for its heat activation. EMBO J 1985;4:2971–6. Mifflin LC, Cohen RE. Characterization of denatured protein inducers of the heat shock (stress) response in Xenopus laevis oocytes. J Biol Chem 1994a;269:15710–7. Mifflin LC, Cohen RE. Hsc70 moderates the heat shock (stress) response in Xenopus laevis oocytes and binds to denatured protein inducers. J Biol Chem 1994b;269:15718–23. Miledi R, Eusebi F, Martinez-Torres A, Palma E, Trettel F. Expression of functional neurotransmitter receptors in Xenopus oocytes after injection of human brain membranes. Proc Natl Acad Sci U S A 2002;99:13238–42. Miledi R, Duenas Z, Martinez-Torres A, Kawas CH, Eusebi F. Microtransplantation of functional receptors and channels from the Alzheimer's brain to frog oocytes. Proc Natl Acad Sci U S A 2004;101:1760–3. Morimoto RI. Regulation of the heat shock transcriptional response: cross talk between a family of heat shock factors, molecular chaperones, and negative regulators. Genes Dev 1998;12: 3788–96. Morrow G, Heikkila JJ, Tanguay RM. Differences in the chaperonelike activities of the four main small heat shock proteins of Drosophila melanogaster. Cell Stress Chaperones 2006;11:51–60. Ovsenek N, Heikkila JJ. DNA sequence specific binding activity of the Xenopus heat shock transcription factor is heat inducible before the midblastula transition. Development 1990;110:427–33. Paine PL, Johnson ME, Lau YT, Tluczek LJ, Miller DS. The oocyte nucleus isolated in oil retains in vivo structure and functions. Biotechniques 1992;13:238–46. Pelham HRB, Bienz M. A synthetic heat shock promoter element confers heat-inducibility on the herpes simplex virus thymidine kinase gene. EMBO J 1982;1:1473–7. Quinlan R, Van Den Ijssel P. Fatal attraction: when chaperones turn harlot. Nat. Med. 1999;5:25–6. Sakai T, Hidehito T, Tenno T, Ito Y, Kokubo T, Hiroaki H, et al. In-cell NMR spectroscopy of proteins inside Xenopus laevis oocytes. J Biomol NMR 2006;36:179–88. Schlatter H, Langer T, Rosmus S, Onneken ML, Fasold H. A novel function for the 90 kDa heat shock protein (Hsp90): facilitating nuclear export of 60 S ribosomal subunits. Biochem J 2002;362: 675–84. Seghal A, Wall DA, Chao MV. Efficient processing and expression of human nerve growth factor receptors in Xenopus laevis: Effects on maturation. Mol Cell Biol 1988;8:2242–6. Selenko P, Server Z, Gadea B, Ruderman J, Wagner G. Quantitative NMR analysis of the protein G B1 domain in Xenopus laevis egg extracts and intact oocytes. Proc Natl Acad Sci U S A 2006;103: 11904–9. Smith GB, Umbach JA, Hirano A, Gundersen CB. Interaction between constitutively expressed heat shock protein, Hsc70, and cysteine string protein is important for cortical granule exocytosis in Xenopus oocytes. J Biol Chem 2005;280:32669–75. Tokmakov AA, Matsumoto E, Shirouzu M, Yokoyama S. Coupled cytoplasmic transcription-and-translation — a method of choice J.J. Heikkila et al. / Biotechnology Advances 25 (2007) 385–395 for heterologous gene expression in Xenopus oocytes. J Biotechnol 2006;122:5–15. Torok Z, Tsvetkova NM, Balogh G, Horvath I, Nagy E, Penzes E, et al. Heat shock protein coinducers with no effect on protein denaturation specifically modulate the membrane lipid phase. Proc Natl Acad Sci U S A 2003;100:3131–6. Tsukahara F, Maru Y. Identification of novel nuclear export and nuclear localization-related signals in human heat shock cognate protein 70. J Biol Chem 2004;279:8867–72. Uzawa M, Grams J, Madden B, Toft D, Salisbury JL. Identification of a complex between centrin and heat shock proteins in CSF-arrested Xenopus oocytes and dissociation of the complex following oocyte activation. Dev Biol 1995;171:51–9. Voellmy R. On mechanisms that control heat shock transcription factor activity in metazoan cells. Cell Stress Chaperones 2004;9:122–33. Voellmy R, Rungger D. Transcription of a Drosophila heat shock gene is heat-induced in Xenopus oocytes. Proc Natl Acad Sci USS 1982;79:1776–80. Voellmy R, Ahmed A, Schiller P, Bromley P, Rungger D. Isolation and functional analysis of a human 70,000-Dalton heat shock protein gene segment. Proc Natl Acad Sci U S A 1985;82: 4949–53. 395 Westwood JT, Clos J, Wu C. Stress-induced oligmerization and chromosomal relocalization of heat shock factor. Nature 1991;353: 822–7. Wormington M. Preparation of synthetic mRNAs and analyses of translational efficiency in microinjected Xenopus oocytes. Xenopus laevis: practical uses in cell and molecular biology. Methods in cell biologySan Diego: Academic Press Inc.; 1991. p. 167–83. Wu C, Wilson S, Walker B, Dawid I, Paisley T, Zimarino V, et al. Purification and properties of Drosophila heat shock activator protein. Science 1987;238:1247–53. Xavier IJ, Mercier PA, McLoughlin CM, Ali A, Woodgett JR, Ovsenek N. Glycogen synthase kinase 3β negatively regulates both DNAbinding and transcriptional activities of heat shock factor 1. J Biol Chem 2000;275:29147–52. Zhou S, Yang SQ, Standring DN. Characterization of Hepatitis B virus capsid particle assembly in Xenopus oocytes. J Virol 1992;66: 3086–92. Zuo J, Baler R, Dahl G, Voellmy R. Activation of the DNA-binding ability of human heat shock transcription factor 1 may involve the transition from an intramolecular to an intermolecular triplestranded coiled-coil structure. Mol Cell Biol 1994;14:7557–68. Zuo J, Rungger D, Voellmy R. Multiple layers of regulation of human heat shock transcription factor 1. Mol Cell Biol 1995;15:4319–30.