Induction of Hsp70 by desiccation, ionising radiation and heat-shock in the eutardigrade Richtersius coronifer

Abstract

The physiology and biochemistry behind the extreme tolerance to desiccation shown by the so-called anhydrobiotic animals represents an exciting challenge to biology. The current knowledge suggests that both carbohydrates and proteins are often involved in protecting the dry cell from damage, or in the repair of induced damage. Tardigrades belong to the most desiccation-tolerant multicellular organisms, but very little research has been reported on the biochemistry behind desiccation tolerance in this group. We quantified the induction of the heat-shock protein Hsp70, a very wide-spread stress protein, in response to desiccation, ionising radiation, and heating, in the anhydrobiotic tardigrade Richtersius coronifer using an immuno-westernblot method. Elevated levels of Hsp70 were recorded after treatment of both heat and ionising radiation, and also in rehydrated tardigrades after a period of desiccation. In contrast, tardigrades in the desiccated (dry) state had reduced Hsp70 levels compared to the non-treated control group. Our results suggest that Hsp70 may be involved in the physiological and biochemical system underlying desiccation (and radiation) tolerance in tardigrades, and that its role may be connected to repair processes after desiccation rather than to biochemical stabilization in the dry state.

Introduction

The mechanisms by which the so-called anhydrobiotic organisms preserve their cells under extreme desiccation have remained a challenge to biology. In most organisms, a severe drying of cells leads to irreversible damage to cellular structures, and eventually to death of the cell and the entire organism. However, anhydrobiotic organisms are apparently able to prevent such damage, or to successfully repair damage that arises. In previous research on the biochemistry of anhydrobiotic organisms, a strong focus has been on the role of polyhydroxy compounds, mainly carbohydrates, as membrane stabilizers in the dry state (see reviews in, e.g., Hoekstra et al., 1997, Crowe, 2002). More recently, a more diversified approach has emerged. In particular, the role of several stress proteins (heat-shock proteins and late embryogenesis proteins) in the protection against desiccation damage has been emphasized (e.g., Goyal et al., 2005, Clegg, 2005). Stress proteins act as molecular chaperones and bind to other proteins, thereby preventing aggregation or unfolding of the protein or promoting protein folding (Ellis and van der Vies, 1991, Gething and Sambrock, 1992, Kiang and Tsokos, 1998, Feder and Hofmann, 1999). Stress proteins may also protect cells from oxidative damage both in vivo and in vitro (Jacquier-Sarlin et al., 1994, Plumier et al., 1995). They may also interact with carbohydrates in protecting against cell damage (Elliott et al., 1996, Singer and Lindquist, 1998, Viner and Clegg, 2001, Ma et al., 2005). Late embryogenesis abundant (LEA) proteins are a complex of proteins originally identified in plant embryos in connection with water stress. Recently, Browne et al. (2002) described the induction of a group 3 LEA protein in the anhydrobiotic nematode Aphelenchus avenae in connection with desiccation. In vitro analyses indicate that this protein may serve a chaperone function, by preventing desiccation-induced aggregation of other proteins, and thus play an important role in the tolerance to desiccation in these animals.

The Hsp70 family is the largest and most conserved group of heat-shock proteins, participating in the folding of newly synthesized and denatured proteins, and inhibiting protein aggregation (e.g., Kiang and Tsokos, 1998, Fink, 1999). There is also evidence that it plays a role in gene regulation by interacting with transcription factors (Wickner et al., 1991). Hsp70 has been shown to interact with both membrane proteins (Fink, 1999), and membrane lipids (Arispe et al., 2002). In extreme desiccation-tolerant (anhydrobiotic) organisms, Hsp70 has not been investigated very much, but its strong connection to general stress and documented association to cell membranes suggests that it may well play a role in the desiccation tolerance machinery.

Tardigrades represent one of the three main invertebrate taxa where anhydrobiotic populations are wide-spread, the other two groups being nematodes and rotifers (Wright et al., 1992). In these taxa, the anhydrobiotic state may be induced over the whole life cycle, from the egg to the adult stage (holo-anhydrobiosis, Jönsson, 2005). The biochemical mechanisms and components behind anhydrobiotic survival in tardigrades are largely unknown, but the eutardigrade Richtersius coronifer was shown to accumulate the disaccharide trehalose at about 2.3% dry weight at the entrance of the anhydrobiotic state (Westh and Ramløv, 1991). Only two previous studies have investigated the induction of stress proteins in tardigrades. Ramløv and Westh (2001) described the appearance of a protein with a molecular mass of 71 kDa from anhydrobiotic specimens of the tardigrade R. coronifer and the absence of the protein from active tardigrades. In a more extensive study, Schill et al. (2004) documented three heat-shock protein (Hsp70 family) genes in the tardigrade species Milnesium tardigradum and their different expressions which are involved in the cycle of dehydration, cryptobiosis and rehydration.

Here we report a study on the induction of Hsp70 in response to desiccation, heat and ionising radiation, in the limno-terrestrial eutardigrade R. coronifer, one of the most well-studied tardigrades with respect to cryptobiosis (e.g., Westh and Ramløv, 1991, Ramløv and Westh, 2001, Jönsson and Rebecchi, 2002, Jönsson et al., 2001). We directly quantified the levels of induced Hsp70 using the immuno-westernblot method.