Different proteins exist to support the stability of animal cells. The intermediate filament proteins are an important example. One type – called lamins – stabilizes the nucleus (the structure within an animal cell that stores most of its DNA), while another forms scaffold-like structures in the rest of cell. The second type, referred to as “cytoplasmic” intermediate filaments, are not found in many hard-bodied creatures including insects and their closest relatives. This is probably because these animals, which are collectively known as arthropods, are instead supported by their tough external skeleton.

The soft-bodied animals called tardigrades (also known as water bears or moss piglets) are closely related to the arthropods. These microscopic animals can endure extreme environmental conditions such as freezing. The tardigrade’s endurance is likely to require some way to stabilize the animal’s cells. This might involve cytoplasmic intermediate filaments, but nothing was known about these proteins in tardigrades.

Now, Hering, Bouameur, Reichelt et al. have investigated if, and where, intermediate filaments are found in the cells of tardigrades. First, the complete set of active genes was analyzed for a species of tardigrade called Hypsibius dujardini; this revealed that three genes for intermediate filament proteins were active. Staining tissue slices or whole tardigrades with a marker that binds to intermediate filament proteins revealed that two of the three proteins were lamins and located within the nucleus. The third protein, which has been named "cytotardin", was found outside of the nucleus. However, unlike well-known cytoplasmic intermediate filaments, this protein did not form scaffold-like structures throughout the cell. Instead, cytotardin formed belt-like filaments that encircled each cell in the skin of the tardigrades.

Hering, Bouameur, Reichelt et al. then discovered that cytotardin seems to be more closely related to lamins than it is to cytoplasmic intermediate filaments. This suggests that cytotardin actually evolved from a tardigrade lamin and then acquired a new role in building filaments outside of the nucleus.

The fact that cytotardin is only found in the skin of the tardigrade and in those tissues that experience mechanical stress (for example, the mouth and legs) hints that it might help stabilize these cells. This could mean that the protein also helps these animals to resist extreme conditions. Further studies should focus on clarifying cytotardin’s role in stabilizing cells, in particular if it is required for the tardigrades' tolerance to environmental stress.

Tardigrades, also known as water bears, are microscopic invertebrates that live in marine, freshwater and semi-aquatic/limno-terrestrial environments (Kinchin, 1994; Nelson, 2002) (Figure 1). Although tardigrades have become renowned for their ability to survive extreme conditions (Møbjerg et al., 2011; Ramløv and Westh, 2001), including exposure to space (Persson et al., 2011; Rebecchi et al., 2011), only little is known about the actual mechanisms that allow their cells to resist severe mechanical stress caused by desiccation and freezing (Møbjerg et al., 2011; Ramløv and Westh, 2001; Wright, 2001; Tanaka et al., 2015; Yamaguchi et al., 2012). The integrity and plasticity of tardigrade tissues might be achieved by specialised cytoskeletal components, such as IF proteins, which are known to be essential for stress resilience of cells (Herrmann et al., 2009; Coulombe and Wong, 2004; Kim and Coulombe, 2007). While lamins, a group of IF proteins found in the nucleus, occur in most eukaryotes, including social amoebae (Krüger et al., 2012) and all metazoans (Dittmer and Misteli, 2011), cytoplasmic IF proteins are thought to have evolved from an ancestral lamin gene by duplication in the bilaterian lineage (Erber et al., 1999; Herrmann and Strelkov, 2011). Genomic and biochemical studies have revealed that the cytoplasmic IF proteins are present in all bilaterian taxa excluding arthropods (Bartnik and Weber, 1989; Goldstein and Gunawardena, 2000; Erber et al., 1998) [but see a contradictory report (Mencarelli et al., 2011) of a putative cytoplasmic IF protein in a collembolan]. The apparent loss of cytoplasmic IF proteins in the arthropod lineage might correlate with the acquisition of an exoskeleton (Herrmann and Strelkov, 2011; Goldstein and Gunawardena, 2000; Erber et al., 1998), which provides mechanical support to the arthropod skin. However, this hypothesis has never been tested, as it is unknown whether or not onychophorans and tardigrades, the soft-bodied relatives of arthropods, possess cytoplasmic IF proteins.

Figure 1

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To clarify whether or not onychophorans and tardigrades possess cytoplasmic IF proteins, we analysed Illumina-sequenced transcriptomes of five distantly related onychophoran species and the freshwater tardigrade H. dujardini. Although this model tardigrade (Gabriel et al., 2007; Gross et al., 2015) shows only a limited ability to tolerate desiccation (anhydrobiosis), it clearly survives immediate freezing (cryobiosis; see Video 1). Our analyses revealed only one putative IF protein transcript in each of the five onychophoran species but three in H. dujardini. Additional screening of the recently sequenced genome (Boothby et al., 2015; Koutsovoulos et al., 2015) of H. dujardini confirms that the identified transcripts correspond to the three potential IF protein-coding genes of this species. According to sequence comparisons with well-characterized IF proteins from humans and the nematode Caenorhabditis elegans, all corresponding proteins of the three identified genes share a similar α-helical rod domain organization with three coiled coil-forming segments (coil 1A, coil 1B, coil 2; Figure 2A,B and Figure 2—figure supplements 1 and 2) (review Chernyatina et al., 2015).This, in conjunction with the highly conserved intermediate filament consensus motifs (review Herrmann and Aebi, 2004) at the beginning and end of the rod domain of all three tardigrade proteins, classify them as intermediate filament proteins. The three tardigrade IF proteins further possess 42 residues in the coil 1B (Figure 2B and Figure 2—figure supplement 1) — a feature that is shared between all eukaryote lamins and protostome cytoplasmic IF proteins but that must have been deleted from the ancestral cytoplasmic IF protein gene in chordates (Herrmann et al., 2009; Peter and Stick, 2015). In contrast to the similar organization of the rod domain, the flanking sequences vary between all three tardigrade IF proteins. One of them, which we named lamin-2, possesses all major regions known from other eukaryote lamins (Herrmann et al., 2009; Dittmer and Misteli, 2011; Burke and Stewart, 2013), including the nuclear localization signal (NLS) — which generally mediates the import of proteins into the nucleus (Mical et al., 2004) — the lamin-tail domain (LTD), and the carboxy-terminal CaaX motif (Figure 2A, B). In contrast, the second tardigrade IF protein (named lamin-1) lacks the CaaX box, whereas the third IF protein from H. dujardini is missing all three motifs, including the NLS, indicating this protein may instead localize in the cytoplasm rather than the nuclear lamina of H. dujardini cells. Since the structure of the latter resembles the domain composition of known bilaterian cytoplasmic IF proteins (Figure 2—figure supplements 1 and 2) we consequently named it cytotardin.

Video 1

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To firmly place the onychophoran and tardigrade IF homologs on the evolutionary tree, we reconstructed the phylogeny of broadly sampled metazoan lamin and cytoplasmic IF protein genes. In our phylogenetic analyses, all identified tardigrade and onychophoran sequences cluster within the bilaterian lamin clade, whereas none of them groups with cytoplasmic IF protein-coding genes (Figure 3 and Figure 3—figure supplements 1 and 2). Surprisingly, the three identified IF sequences of H. dujardini, together with two sequences from Milnesium tardigradum, form a strongly supported monophyletic clade of tardigrade lamins (GTR+G: Bootstrap support BS=77, LG+G: BS=80) in our analyses (Figure 3 and Figure 3—figure supplements 1 and 2). This implies that there were at least two duplication events in the tardigrade lineage that gave rise to lamin-1, lamin-2 and cytotardin genes — consequently characterizing the tardigrade IFs (including cytotardin), for example, as co-orthologous to nematode lamins rather than orthologous to nematode cytoplasmic IFs (Figure 3 and Figure 3—figure supplements 1 and 2). Our results further show that the isomin sequence of the collembolan Isotomurus maculatus does in fact cluster with other identified collembolan transcripts (GTR+G: BS=74, LG+G: BS=70) within the clade of arthropod lamins (Figure 3 and Figure 3—figure supplements 1 and 2); it had previously been interpreted (Mencarelli et al., 2011) as closely related to cytoplasmic IF proteins of nematodes and therefore as an ortholog of the bilaterian cytoplasmic IF proteins, likely due to the narrower dataset used for their phylogenetic analysis. These results clearly challenge the identity of isomin as a member of the bilaterian cytoplasmic IF protein clade (Mencarelli et al., 2011) and suggest that orthologs of genes encoding these proteins are entirely missing in arthropods, at least in those with known genomic sequences. In fact, besides the putative IF proteins from chelicerates, crustaceans and hexapods obtained from publicly available databases (e.g. GenBank), our transcriptomic and genomic analyses, which included screening of the genome of the centipede Strigamia maritima (see Chipman et al., 2014), the water flea Daphnia pulex (see Colbourne et al., 2011), and more than 70 transcriptomes from hexapod species sequenced as part of the 1KITE project (Misof et al., 2014), strongly suggest that these genes were already lost in the panarthropod lineage, since all of these panarthropod IF proteins cluster within a well-supported monophyletic clade of bilaterian lamins (GTR+G: BS=84, LG+G: BS=76; Figure 3—figure supplements 1 and 2). In this respect, even if the metazoan lamins are polyphyletic, as recently proposed by Kollmar, 2015 based on the finding of putative nematocilin homologs in Bilateria, our results favour the tardigrade, copepod, and collembolan IF proteins as members of the bilaterian lamins rather than the bilaterian cytoplasmic IFs or nematocilins (Figure 3—figure supplements 1 and 2).

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To determine their subcellular localization and organization, we generated antisera against the three IF proteins of H. dujardini and confirmed their specificity (see Figure 4—figure supplement 1 and Figure 5—figure supplement 1). Immunolocalization of lamin-1, lamin-2 and cytotardin proteins in whole-mount preparations and cryosectioned specimens, in conjunction with confocal laser-scanning microscopy, revealed a highly specific subcellular distribution and tissue-restricted expression of these proteins in H. dujardini. As predicted, lamin-1 and lamin-2 proteins both display a typical, lamin-like distribution within all nuclei (Figure 4A–D and Figure 4—figure supplement 2). While lamin-1 is localised throughout the nucleoplasm (Figure 4A), lamin-2 is restricted to the nuclear periphery (Figure 4B), although there is a small overlap region between these two proteins (Figure 4C). The intranuclear distribution of lamin-1 corresponds to its lack of the CaaX motif, which is responsible for the association of lamins with the nuclear envelope (Kitten and Nigg, 1991). In contrast to the two lamins, cytotardin of H. dujardini is not localised within the nucleus, but in the peripheral cytoplasm of all epidermal and foregut cells, where it appears to be closely associated or aligned with the plasma membrane (Figure 4E–G and Figure 4—figure supplement 3 and Figure 6—figure supplement 1). In this setting, it encircles the apical regions of the cells in a belt-like, filamentous array (Figure 4E–G and Figure 4—figure supplement 3).

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In order to investigate the filament-forming capacity of cytotardin, we transiently transfected human mammary epithelial MCF-7 cells with a corresponding cDNA (Figure 5A–F and Figure 5—figure supplement 1). Immunofluorescence analyses using cytotardin antibody revealed that this protein is located exclusively in the cytoplasm of transfected MCF-7 cells, where it forms both short filaments and extensive cytoskeletal networks which most likely are homopolymeric (Figure 5A–F). Notably, some of the cytotardin arrays display cage-like perinuclear structures, while others are located in the periphery close to the cell membrane (Figure 5E,F). Double labelling for cytotardin and desmoplakin, a desmosomal protein mediating membrane attachment of mammalian IF proteins (Simpson et al., 2011), shows that cytotardin occurs close to desmosomes but is not co-localised with desmoplakin (Figure 5C,D). To examine whether this arrangement was mediated by interactions between cytotardin and keratins endogenously expressed in MCF-7 cells, we double-labelled the transfectants for cytotardin and keratin-8. Our data show that these two proteins are not co-localised and that the endogenous keratin networks are displaced in cells with dense cytotardin arrays (Figure 5E,F). These findings strongly support an intrinsic ability of the cytotardin protein of H. dujardini to both form homopolymeric filaments and cytoplasmic networks — both properties that are functionally analogous to mammalian cytoplasmic IFs (Bohnekamp et al., 2015).

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Our data on tissue-specific distribution of cytotardin in H. dujardini further show that its belt-like arrays are confined to the ectodermal epithelia, including the epidermis, buccal tube, pharynx, and oesophagus (Figures 4E–G and 6A,B and Figure 4—figure supplement 3 and Figure 6—figure supplement 1). Thus, the entire tardigrade body is ensheathed by a grid of belt-like filaments formed by the cytotardin protein, which retain their integrity even in contracted specimens (Figure 4E–G and Figure 4—figure supplement 3 and Figure 6—figure supplement 1). The most prominent anti-cytotardin immunoreactivity is found in areas exposed to considerable physical stress in living specimens, including the bases of claws and the stylet apparatus (Figures 4F,G and 6B and Figure 4—figure supplement 3 and Figure 6—figure supplement 1). We therefore anticipate that much of the resistance of the tardigrade body to extreme conditions, such as cryobiosis (Wright, 2001; Hengherr et al., 2009), might be attributable to the dense, fibre-like cytotardin meshwork. The belt-like structures encircling each epidermal cell might help to resist the shearing forces that arise during freezing and thawing cycles, whereas the dense meshwork at the basis of each claw and around the stylets might provide the tissue stability necessary for locomotion and feeding.

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Together, our data demonstrate that cytotardin of H. dujardini is a cytoplasmic IF protein, predominantly expressed in ectodermal epithelia, that has evolved independently from the cytoplasmic IF proteins of other bilaterians by gene duplication and subsequent neofunctionalization (Figure 6C). This process was most likely triggered by an initial loss of the CaaX motif and nuclear localization signal from the ancestral lamin gene in the tardigrade lineage. Expression of the cytotardin protein in tissues that are subject to mechanical stress might have served as a pre-adaptation for the ability of tardigrades to survive extreme environmental conditions. Similar duplication and neofunctionalization events might have occurred in the collembolan and copepod lineages, as our findings demonstrate that these two taxa also show duplicated lamins that have lost their nuclear localization signals (Figures 3 and 6C and Figure 3—figure supplements 1 and 2). While isomin might stabilize the intestinal epithelial cells in collembolans (Mencarelli et al., 2011), the localization and function of the putative lamin-derived cytoplasmic IF protein in copepods has yet to be clarified.

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Author details

1. Lars Hering

   1. Department of Zoology, Institute of Biology, University of Kassel, Kassel, Germany
   2. Animal Evolution and Development, Institute of Biology, University of Leipzig, Leipzig, Germany

   Contribution

   LH, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Contributed equally with

   Jamal-Eddine Bouameur and Julian Reichelt

   For correspondence

   lars.hering@uni-kassel.de

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0003-4469-991X

2. Jamal-Eddine Bouameur

   Institute of Biology and Translational Center for Regenerative Medicine, University of Leipzig, Leipzig, Germany

   Contribution

   J-EB, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Contributed equally with

   Lars Hering and Julian Reichelt

   Competing interests

   The authors declare that no competing interests exist.

3. Julian Reichelt

   Department of Zoology, Institute of Biology, University of Kassel, Kassel, Germany

   Contribution

   JR, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Contributed equally with

   Lars Hering and Jamal-Eddine Bouameur

   Competing interests

   The authors declare that no competing interests exist.

4. Thomas M Magin

   Institute of Biology and Translational Center for Regenerative Medicine, University of Leipzig, Leipzig, Germany

   Contribution

   TMM, Conception and design, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

5. Georg Mayer

   1. Department of Zoology, Institute of Biology, University of Kassel, Kassel, Germany
   2. Animal Evolution and Development, Institute of Biology, University of Leipzig, Leipzig, Germany

   Contribution

   GM, Conception and design, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   georg.mayer@uni-kassel.de

   Competing interests

   The authors declare that no competing interests exist.

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