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.

1. 1. Hering L
   2. Bouameur J-E
   3. Reichelt J
   4. Magin TM
   5. Mayer G

   (2016) Hypsibius dujardini cytotardin

   Publicly available at the NCBI GenBank (accession no. KU295462).

   http://www.ncbi.nlm.nih.gov/nuccore/KU295462
2. 1. Hering L
   2. Bouameur J-E
   3. Reichelt J
   4. Magin TM
   5. Mayer G

   (2016) Hypsibius dujardini lamin-1

   Publicly available at the NCBI GenBank (accession no. KU295460).

   http://www.ncbi.nlm.nih.gov/nuccore/KU295460
3. 1. Hering L
   2. Bouameur J-E
   3. Reichelt J
   4. Magin TM
   5. Mayer G

   (2016) Hypsibius dujardini lamin-2

   Publicly available at the NCBI GenBank (accession no. KU295461).

   http://www.ncbi.nlm.nih.gov/nuccore/KU295461
4. 1. Hering L
   2. Bouameur J-E
   3. Reichelt J
   4. Magin TM
   5. Mayer G

   (2016) Euperipatoides rowelli lamin

   Publicly available at the NCBI GenBank (accession no. KU295463).

   http://www.ncbi.nlm.nih.gov/nuccore/KU295463
5. 1. Hering L
   2. Bouameur J-E
   3. Reichelt J
   4. Magin TM
   5. Mayer G

   (2016) Phallocephale tallagandensis lamin

   Publicly available at the NCBI GenBank (accession no. KU295464).

   http://www.ncbi.nlm.nih.gov/nuccore/KU295464
6. 1. Hering L
   2. Bouameur J-E
   3. Reichelt J
   4. Magin TM
   5. Mayer G

   (2016) Ooperipatus hispidus lamin

   Publicly available at the NCBI GenBank (accession no. KU295465).

   http://www.ncbi.nlm.nih.gov/nuccore/KU295465
7. 1. Hering L
   2. Bouameur J-E
   3. Reichelt J
   4. Magin TM
   5. Mayer G

   (2016) Principapillatus hitoyensis lamin

   Publicly available at the NCBI GenBank (accession no. KU295466).

   http://www.ncbi.nlm.nih.gov/nuccore/KU295466
8. 1. Hering L
   2. Bouameur J-E
   3. Reichelt J
   4. Magin TM
   5. Mayer G

   (2016) Eoperipatus sp. LH-2012 lamin

   Publicly available at the NCBI GenBank (accession no. KU295467).

   http://www.ncbi.nlm.nih.gov/nuccore/KU295467

1. 1. Koutsovoulos G
   2. Kumar S
   3. Laetsch DR
   4. Stevens L
   5. Daub J
   6. Conlon C
   7. Marhoon H
   8. Thomas F
   9. Aboobaker A
   10. Blaxter M

   (2015) Data from: The genome of the tardigrade Hypsibius dujardini (doi: 10.1101/033464)

   Publicly available at http://badger.bio.ed.ac.uk/H_dujardini/.

   http://badger.bio.ed.ac.uk/H_dujardini/
2. 1. Boothby TC
   2. Tenlen JR
   3. Smith FW
   4. Wang JR
   5. Patanella KA
   6. Osborne Nishimura E
   7. Tintori SC
   8. Li Q
   9. Jones CD
   10. Yandell M
   11. Messina DN
   12. Glasscock J
   13. Goldstein B

   (2015) Data from: Evidence for extensive horizontal gene transfer from the draft genome of a tardigrade (doi: 10.1073/pnas.1510461112)

   Publicly available at http://weatherby.genetics.utah.edu/seq_transf/.

   http://weatherby.genetics.utah.edu/seq_transf/
3. 1. Chipman AD
   2. et al.

   (2014) Data from: The first myriapod genome sequence reveals conservative arthropod gene content and genome organisation in the centipede Strigamia maritima (doi: 10.1371/journal.pbio.1002005)

   Publicly available at http://metazoa.ensembl.org/Strigamia_maritima.

   http://metazoa.ensembl.org/Strigamia_maritima
4. 1. Adamski M
   2. Leininger S
   3. Bergum B
   4. Liu J
   5. Adamska K

   (2013) Protein translations from Sycon ciliatum transcriptome (SCIL_T-PEP_130802)

   Publicly available at the Compagen platform for early branching metazoan animals (http://www.compagen.org/datasets.html).

   http://www.compagen.org/datasets/SCIL_T-PEP_130802
5. 1. Richter DJ
   2. Mora J
   3. Nichols SA

   (2013) Protein translations from Oscarella carmela transcriptome (OCAR_T-PEP_130911)

   Publicly available at the Compagen platform for early branching metazoan animals (http://www.compagen.org/datasets.html).

   http://www.compagen.org/datasets/OCAR_T-PEP_130911
6. 1. Authors

   (2012) Oscarella sp. transcriptome (Osp._T-CDS_120614)

   Publicly available at the Compagen platform for early branching metazoan animals (http://www.compagen.org/datasets.html).

   http://www.compagen.org/datasets/Osp._T-CDS_120614
7. 1. Lapébie P
   2. Ruggiero A
   3. Chevalier S
   4. Chang P
   5. Momose T

   (2014) Data from: Differential Responses to Wnt and PCP Disruption Predict Expression and Developmental Function of Conserved and Novel Genes in a Cnidarian (doi: 10.1371/journal.pgen.1004590)

   Publicly available at the Compagen platform for early branching metazoan animals (http://www.compagen.org/datasets.html).

   http://www.compagen.org/datasets/CHEM_T-CDS_141022
8. 1. Moroz LL
   2. Williams PL
   3. Kohn A

   (2013) Bolinopsis infundibulum transcriptome (Bolinopsis_infundibulum_Illumina_RNA-Seq)

   Publicly available at NeuroBase: A Comparative Neurogenomics Database (http://neurobase.rc.ufl.edu/pleurobrachia/browse).

   http://neurobase.rc.ufl.edu/pleurobrachia/browse
9. 1. Moroz LL
   2. Williams PL
   3. Kohn A

   (2015) Beroe abyssicola transcriptome (Beroe_abyssicola_Illumina_RNA-Seq)

   Publicly available at NeuroBase: A Comparative Neurogenomics Database (http://neurobase.rc.ufl.edu/pleurobrachia/browse).

   http://neurobase.rc.ufl.edu/pleurobrachia/browse

1. 1. Akaike H

   (1974) A new look at the statistical model identification

   IEEE Transactions on Automatic Control 19:716–723.

   https://doi.org/10.1109/TAC.1974.1100705

   • Google Scholar
2. 1. Altschul SF
   2. Madden TL
   3. Schäffer AA
   4. Zhang J
   5. Zhang Z
   6. Miller W
   7. Lipman DJ

   (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs

   Nucleic Acids Research 25:3389–3402.

   https://doi.org/10.1093/nar/25.17.3389

   • Google Scholar
3. 1. Bartnik E
   2. Weber K

   (1989)

   Widespread occurrence of intermediate filaments in invertebrates; common principles ans aspects of diversion

   European Journal of Cell Biology 50:17–33.

   • Google Scholar
4. 1. Bohnekamp J
   2. Cryderman DE
   3. Paululat A
   4. Baccam GC
   5. Wallrath LL
   6. Magin TM

   (2015) A Drosophila model of epidermolysis bullosa simplex

   Journal of Investigative Dermatology 135:2031–2039.

   https://doi.org/10.1038/jid.2015.129

   • Google Scholar
5. 1. Boothby TC
   2. Tenlen JR
   3. Smith FW
   4. Wang JR
   5. Patanella KA
   6. Osborne Nishimura E
   7. Tintori SC
   8. Li Q
   9. Jones CD
   10. Yandell M
   11. Messina DN
   12. Glasscock J
   13. Goldstein B

   (2015) Evidence for extensive horizontal gene transfer from the draft genome of a tardigrade

   Proceedings of the National Academy of Sciences of the United States of America 112:15976–15981.

   https://doi.org/10.1073/pnas.1510461112

   • Google Scholar
6. 1. Brameier M
   2. Krings A
   3. MacCallum RM

   (2007) NucPred—predicting nuclear localization of proteins

   Bioinformatics 23:1159–1160.

   https://doi.org/10.1093/bioinformatics/btm066

   • Google Scholar
7. 1. Burke B
   2. Stewart CL

   (2013) The nuclear lamins: flexibility in function

   Nature Reviews Molecular Cell Biology 14:13–24.

   https://doi.org/10.1038/nrm3488

   • Google Scholar
8. 1. Chernyatina AA
   2. Guzenko D
   3. Strelkov SV

   (2015) Intermediate filament structure: the bottom-up approach

   Current Opinion in Cell Biology 32:65–72.

   https://doi.org/10.1016/j.ceb.2014.12.007

   • Google Scholar
9. 1. Chipman AD
   2. Ferrier DEK
   3. Brena C
   4. Qu J
   5. Hughes DST
   6. Schröder R
   7. Torres-Oliva M
   8. Znassi N
   9. Jiang H
   10. Almeida FC
   11. Alonso CR
   12. Apostolou Z
   13. Aqrawi P
   14. Arthur W
   15. Barna JCJ
   16. Blankenburg KP
   17. Brites D
   18. Capella-Gutiérrez S
   19. Coyle M
   20. Dearden PK
   21. Du Pasquier L
   22. Duncan EJ
   23. Ebert D
   24. Eibner C
   25. Erikson G
   26. Evans PD
   27. Extavour CG
   28. Francisco L
   29. Gabaldón T
   30. Gillis WJ
   31. Goodwin-Horn EA
   32. Green JE
   33. Griffiths-Jones S
   34. Grimmelikhuijzen CJP
   35. Gubbala S
   36. Guigó R
   37. Han Y
   38. Hauser F
   39. Havlak P
   40. Hayden L
   41. Helbing S
   42. Holder M
   43. Hui JHL
   44. Hunn JP
   45. Hunnekuhl VS
   46. Jackson L
   47. Javaid M
   48. Jhangiani SN
   49. Jiggins FM
   50. Jones TE
   51. Kaiser TS
   52. Kalra D
   53. Kenny NJ
   54. Korchina V
   55. Kovar CL
   56. Kraus FB
   57. Lapraz F
   58. Lee SL
   59. Lv J
   60. Mandapat C
   61. Manning G
   62. Mariotti M
   63. Mata R
   64. Mathew T
   65. Neumann T
   66. Newsham I
   67. Ngo DN
   68. Ninova M
   69. Okwuonu G
   70. Ongeri F
   71. Palmer WJ
   72. Patil S
   73. Patraquim P
   74. Pham C
   75. Pu L-L
   76. Putman NH
   77. Rabouille C
   78. Ramos OM
   79. Rhodes AC
   80. Robertson HE
   81. Robertson HM
   82. Ronshaugen M
   83. Rozas J
   84. Saada N
   85. Sánchez-Gracia A
   86. Scherer SE
   87. Schurko AM
   88. Siggens KW
   89. Simmons D
   90. Stief A
   91. Stolle E
   92. Telford MJ
   93. Tessmar-Raible K
   94. Thornton R
   95. van der Zee M
   96. von Haeseler A
   97. Williams JM
   98. Willis JH
   99. Wu Y
   100. Zou X
   101. Lawson D
   102. Muzny DM
   103. Worley KC
   104. Gibbs RA
   105. Akam M
   106. Richards S

   (2014) The first myriapod genome sequence reveals conservative arthropod gene content and genome organisation in the centipede Strigamia maritima

   PLoS Biology 12:e1002005.

   https://doi.org/10.1371/journal.pbio.1002005

   • Google Scholar
10. 1. Colbourne JK
    2. Pfrender ME
    3. Gilbert D
    4. Thomas WK
    5. Tucker A
    6. Oakley TH
    7. Tokishita S
    8. Aerts A
    9. Arnold GJ
    10. Basu MK
    11. Bauer DJ
    12. Cáceres CE
    13. Carmel L
    14. Casola C
    15. Choi J-H
    16. Detter JC
    17. Dong Q
    18. Dusheyko S
    19. Eads BD
    20. Fröhlich T
    21. Geiler-Samerotte KA
    22. Gerlach D
    23. Hatcher P
    24. Jogdeo S
    25. Krijgsveld J
    26. Kriventseva EV
    27. Kültz D
    28. Laforsch C
    29. Lindquist E
    30. Lopez J
    31. Manak JR
    32. Muller J
    33. Pangilinan J
    34. Patwardhan RP
    35. Pitluck S
    36. Pritham EJ
    37. Rechtsteiner A
    38. Rho M
    39. Rogozin IB
    40. Sakarya O
    41. Salamov A
    42. Schaack S
    43. Shapiro H
    44. Shiga Y
    45. Skalitzky C
    46. Smith Z
    47. Souvorov A
    48. Sung W
    49. Tang Z
    50. Tsuchiya D
    51. Tu H
    52. Vos H
    53. Wang M
    54. Wolf YI
    55. Yamagata H
    56. Yamada T
    57. Ye Y
    58. Shaw JR
    59. Andrews J
    60. Crease TJ
    61. Tang H
    62. Lucas SM
    63. Robertson HM
    64. Bork P
    65. Koonin EV
    66. Zdobnov EM
    67. Grigoriev IV
    68. Lynch M
    69. Boore JL

    (2011) The ecoresponsive genome of Daphnia pulex

    Science 331:555–561.

    https://doi.org/10.1126/science.1197761

    • Google Scholar
11. 1. Cole C
    2. Barber JD
    3. Barton GJ

    (2008) The Jpred 3 secondary structure prediction server

    Nucleic Acids Research 36:W197–W201.

    https://doi.org/10.1093/nar/gkn238

    • Google Scholar
12. 1. Coulombe PA
    2. Wong P

    (2004) Cytoplasmic intermediate filaments revealed as dynamic and multipurpose scaffolds

    Nature Cell Biology 6:699–706.

    https://doi.org/10.1038/ncb0804-699

    • Google Scholar
13. 1. Darriba D
    2. Taboada GL
    3. Doallo R
    4. Posada D

    (2011) ProtTest 3: fast selection of best-fit models of protein evolution

    Bioinformatics 27:1164–1165.

    https://doi.org/10.1093/bioinformatics/btr088

    • Google Scholar
14. 1. Dittmer TA
    2. Misteli T

    (2011) The lamin protein family

    Genome Biology 12:222.

    https://doi.org/10.1186/gb-2011-12-5-222

    • Google Scholar
15. 1. Dress AWM
    2. Flamm C
    3. Fritzsch G
    4. Grünewald S
    5. Kruspe M
    6. Prohaska SJ
    7. Stadler PF

    (2008) Noisy: identification of problematic columns in multiple sequence alignments

    Algorithms for Molecular Biology 3:7.

    https://doi.org/10.1186/1748-7188-3-7

    • Google Scholar
16. 1. Erber A
    2. Riemer D
    3. Bovenschulte M
    4. Weber K

    (1998) Molecular phylogeny of metazoan intermediate filament proteins

    Journal of Molecular Evolution 47:751–762.

    https://doi.org/10.1007/PL00006434

    • Google Scholar
17. 1. Erber A
    2. Riemer D
    3. Hofemeister H
    4. Bovenschulte M
    5. Stick R
    6. Panopoulou G
    7. Lehrach H
    8. Weber K

    (1999) Characterization of the Hydra lamin and its gene: a molecular phylogeny of metazoan lamins

    Journal of Molecular Evolution 49:260–271.

    https://doi.org/10.1007/PL00006548

    • Google Scholar
18. 1. Finn RD
    2. Bateman A
    3. Clements J
    4. Coggill P
    5. Eberhardt RY
    6. Eddy SR
    7. Heger A
    8. Hetherington K
    9. Holm L
    10. Mistry J
    11. Sonnhammer ELL
    12. Tate J
    13. Punta M

    (2014) Pfam: the protein families database

    Nucleic Acids Research 42:D222–D230.

    https://doi.org/10.1093/nar/gkt1223

    • Google Scholar
19. 1. Gabriel WN
    2. McNuff R
    3. Patel SK
    4. Gregory TR
    5. Jeck WR
    6. Jones CD
    7. Goldstein B

    (2007) The tardigrade Hypsibius dujardini, a new model for studying the evolution of development

    Developmental Biology 312:545–559.

    https://doi.org/10.1016/j.ydbio.2007.09.055

    • Google Scholar
20. 1. Goldstein LSB
    2. Gunawardena S

    (2000) Flying through the Drosophila cytoskeletal genome

    The Journal of Cell Biology 150:F63–F68.

    https://doi.org/10.1083/jcb.150.2.F63

    • Google Scholar
21. Book

    1. Gross V
    2. Treffkorn S
    3. Mayer G

    (2015) Tardigrada

    In: Wanninger A,, editors. Evolutionary Developmental Biology of Invertebrates 3. Vienna: Springer. pp. 35–52.

    https://doi.org/10.1007/978-3-7091-1865-8_3

    • Google Scholar
22. 1. Hemmrich G
    2. Bosch TCG

    (2008) Compagen, a comparative genomics platform for early branching metazoan animals, reveals early origins of genes regulating stem-cell differentiation

    BioEssays 30:1010–1018.

    https://doi.org/10.1002/bies.20813

    • Google Scholar
23. 1. Hengherr S
    2. Worland MR
    3. Reuner A
    4. Brummer F
    5. Schill RO

    (2009) Freeze tolerance, supercooling points and ice formation: comparative studies on the subzero temperature survival of limno-terrestrial tardigrades

    Journal of Experimental Biology 212:802–807.

    https://doi.org/10.1242/jeb.025973

    • Google Scholar
24. 1. Hering L
    2. Henze MJ
    3. Kohler M
    4. Kelber A
    5. Bleidorn C
    6. Leschke M
    7. Nickel B
    8. Meyer M
    9. Kircher M
    10. Sunnucks P
    11. Mayer G

    (2012) Opsins in onychophora (velvet worms) suggest a single origin and subsequent diversification of visual pigments in arthropods

    Molecular Biology and Evolution 29:3451–3458.

    https://doi.org/10.1093/molbev/mss148

    • Google Scholar
25. 1. Hering L
    2. Mayer G

    (2014) Analysis of the opsin repertoire in the tardigrade Hypsibius dujardini provides insights into the evolution of opsin genes in Panarthropoda

    Genome Biology and Evolution 6:2380–2391.

    https://doi.org/10.1093/gbe/evu193

    • Google Scholar
26. 1. Herrmann H
    2. Aebi U

    (2004) Intermediate filaments: molecular structure, assembly mechanism, and integration into functionally distinct intracellular scaffolds

    Annual Review of Biochemistry 73:749–789.

    https://doi.org/10.1146/annurev.biochem.73.011303.073823

    • Google Scholar
27. 1. Herrmann H
    2. Strelkov SV
    3. Burkhard P
    4. Aebi U

    (2009) Intermediate filaments: primary determinants of cell architecture and plasticity

    Journal of Clinical Investigation 119:1772–1783.

    https://doi.org/10.1172/JCI38214

    • Google Scholar
28. 1. Herrmann H
    2. Strelkov SV

    (2011) History and phylogeny of intermediate filaments: now in insects

    BMC Biology 9:.

    https://doi.org/10.1186/1741-7007-9-16

    • Google Scholar
29. 1. Hurvich CM
    2. Tsai C-L

    (1989) Regression and time series model selection in small samples

    Biometrika 76:297–307.

    https://doi.org/10.1093/biomet/76.2.297

    • Google Scholar
30. 1. Katoh K
    2. Standley DM

    (2013) MAFFT multiple sequence alignment software version 7: improvements in performance and usability

    Molecular Biology and Evolution 30:772–780.

    https://doi.org/10.1093/molbev/mst010

    • Google Scholar
31. 1. Kim S
    2. Coulombe PA

    (2007) Intermediate filament scaffolds fulfill mechanical, organizational, and signaling functions in the cytoplasm

    Genes & Development 21:1581–1597.

    https://doi.org/10.1101/gad.1552107

    • Google Scholar
32. Book

    1. Kinchin IM

    (1994)

    The Biology of Tardigrades

    London: Portland Press Inc.

    • Google Scholar
33. 1. Kitten GT
    2. Nigg EA

    (1991) The CaaX motif is required for isoprenylation, carboxyl methylation, and nuclear membrane association of lamin B2

    The Journal of Cell Biology 113:13–23.

    https://doi.org/10.1083/jcb.113.1.13

    • Google Scholar
34. 1. Kollmar M

    (2015) Polyphyly of nuclear lamin genes indicates an early eukaryotic origin of the metazoan-type intermediate filament proteins

    Scientific Reports 5:10652.

    https://doi.org/10.1038/srep10652

    • Google Scholar
35. 1. Kosugi S
    2. Hasebe M
    3. Tomita M
    4. Yanagawa H

    (2009) Systematic identification of cell cycle-dependent yeast nucleocytoplasmic shuttling proteins by prediction of composite motifs

    Proceedings of the National Academy of Sciences of the United States of America 106:10171–10176.

    https://doi.org/10.1073/pnas.0900604106

    • Google Scholar
36. 1. Koutsovoulos G
    2. Kumar S
    3. Laetsch DR
    4. Stevens L
    5. Daub J
    6. Conlon C
    7. Maroon H
    8. Thomas F
    9. Aboobaker A
    10. Blaxter M

    (2015) The genome of the tardigrade Hypsibius dujardini

    bioRxiv.

    https://doi.org/10.1101/033464

    • Google Scholar
37. 1. Krüger A
    2. Batsios P
    3. Baumann O
    4. Luckert E
    5. Schwarz H
    6. Stick R
    7. Meyer I
    8. Graf R

    (2012) Characterization of NE81, the first lamin-like nucleoskeleton protein in a unicellular organism

    Molecular Biology of the Cell 23:360–370.

    https://doi.org/10.1091/mbc.E11-07-0595

    • Google Scholar
38. 1. Letunic I
    2. Bork P

    (2011) Interactive Tree of Life v2: online annotation and display of phylogenetic trees made easy

    Nucleic Acids Research 39:W475–W478.

    https://doi.org/10.1093/nar/gkr201

    • Google Scholar
39. 1. Letunic I
    2. Doerks T
    3. Bork P

    (2015) SMART: recent updates, new developments and status in 2015

    Nucleic Acids Research 43:D257–D260.

    https://doi.org/10.1093/nar/gku949

    • Google Scholar
40. 1. Löffek S
    2. Wöll S
    3. Höhfeld J
    4. Leube RE
    5. Has C
    6. Bruckner-Tuderman L
    7. Magin TM

    (2010) The ubiquitin ligase CHIP/STUB1 targets mutant keratins for degradation

    Human Mutation 31:466–476.

    https://doi.org/10.1002/humu.21222

    • Google Scholar
41. 1. Mayer G
    2. Hering L
    3. Stosch JM
    4. Stevenson PA
    5. Dircksen H

    (2015) Evolution of pigment-dispersing factor neuropeptides in Panarthropoda: insights from Onychophora (velvet worms) and Tardigrada (water bears)

    Journal of Comparative Neurology 523:1865–1885.

    https://doi.org/10.1002/cne.23767

    • Google Scholar
42. 1. McWilliam H
    2. Li W
    3. Uludag M
    4. Squizzato S
    5. Park YM
    6. Buso N
    7. Cowley AP
    8. Lopez R

    (2013) Analysis tool web services from the EMBL-EBI

    Nucleic Acids Research 41:W597–W600.

    https://doi.org/10.1093/nar/gkt376

    • Google Scholar
43. 1. Mencarelli C
    2. Ciolfi S
    3. Caroti D
    4. Lupetti P
    5. Dallai R

    (2011) Isomin: a novel cytoplasmic intermediate filament protein from an arthropod species

    BMC Biology 9:17.

    https://doi.org/10.1186/1741-7007-9-17

    • Google Scholar
44. 1. Mical TI
    2. Luther PW
    3. Monteiro MJ

    (2004) Intracellular assembly and sorting of intermediate filament proteins: role of the 42 amino acid lamin insert

    Experimental Cell Research 295:183–193.

    https://doi.org/10.1016/j.yexcr.2004.01.006

    • Google Scholar
45. 1. Minin V
    2. Abdo Z
    3. Joyce P
    4. Sullivan J

    (2003) Performance-based selection of likelihood models for phylogeny estimation

    Systematic Biology 52:674–683.

    https://doi.org/10.1080/10635150390235494

    • Google Scholar
46. 1. Misof B
    2. Liu S
    3. Meusemann K
    4. Peters RS
    5. Donath A
    6. Mayer C
    7. Frandsen PB
    8. Ware J
    9. Flouri T
    10. Beutel RG
    11. Niehuis O
    12. Petersen M
    13. Izquierdo-Carrasco F
    14. Wappler T
    15. Rust J
    16. Aberer AJ
    17. Aspock U
    18. Aspock H
    19. Bartel D
    20. Blanke A
    21. Berger S
    22. Bohm A
    23. Buckley TR
    24. Calcott B
    25. Chen J
    26. Friedrich F
    27. Fukui M
    28. Fujita M
    29. Greve C
    30. Grobe P
    31. Gu S
    32. Huang Y
    33. Jermiin LS
    34. Kawahara AY
    35. Krogmann L
    36. Kubiak M
    37. Lanfear R
    38. Letsch H
    39. Li Y
    40. Li Z
    41. Li J
    42. Lu H
    43. Machida R
    44. Mashimo Y
    45. Kapli P
    46. McKenna DD
    47. Meng G
    48. Nakagaki Y
    49. Navarrete-Heredia JL
    50. Ott M
    51. Ou Y
    52. Pass G
    53. Podsiadlowski L
    54. Pohl H
    55. von Reumont BM
    56. Schutte K
    57. Sekiya K
    58. Shimizu S
    59. Slipinski A
    60. Stamatakis A
    61. Song W
    62. Su X
    63. Szucsich NU
    64. Tan M
    65. Tan X
    66. Tang M
    67. Tang J
    68. Timelthaler G
    69. Tomizuka S
    70. Trautwein M
    71. Tong X
    72. Uchifune T
    73. Walzl MG
    74. Wiegmann BM
    75. Wilbrandt J
    76. Wipfler B
    77. Wong TKF
    78. Wu Q
    79. Wu G
    80. Xie Y
    81. Yang S
    82. Yang Q
    83. Yeates DK
    84. Yoshizawa K
    85. Zhang Q
    86. Zhang R
    87. Zhang W
    88. Zhang Y
    89. Zhao J
    90. Zhou C
    91. Zhou L
    92. Ziesmann T
    93. Zou S
    94. Li Y
    95. Xu X
    96. Zhang Y
    97. Yang H
    98. Wang J
    99. Wang J
    100. Kjer KM
    101. Zhou X

    (2014) Phylogenomics resolves the timing and pattern of insect evolution

    Science 346:763–767.

    https://doi.org/10.1126/science.1257570

    • Google Scholar
47. 1. Møbjerg N
    2. Halberg KA
    3. Jørgensen A
    4. Persson D
    5. Bjørn M
    6. Ramløv H
    7. Kristensen RM

    (2011) Survival in extreme environments - on the current knowledge of adaptations in tardigrades

    Acta Physiologica 202:409–420.

    https://doi.org/10.1111/j.1748-1716.2011.02252.x

    • Google Scholar
48. 1. Nelson DR

    (2002) Current status of the Tardigrada: evolution and ecology

    Integrative and Comparative Biology 42:652–659.

    https://doi.org/10.1093/icb/42.3.652

    • Google Scholar
49. 1. Persson D
    2. Halberg KA
    3. Jørgensen A
    4. Ricci C
    5. Møbjerg N
    6. Kristensen RM

    (2011) Extreme stress tolerance in tardigrades: surviving space conditions in low earth orbit

    Journal of Zoological Systematics and Evolutionary Research 49:90–97.

    https://doi.org/10.1111/j.1439-0469.2010.00605.x

    • Google Scholar
50. 1. Peter A
    2. Stick R

    (2015) Evolutionary aspects in intermediate filament proteins

    Current Opinion in Cell Biology 32:48–55.

    https://doi.org/10.1016/j.ceb.2014.12.009

    • Google Scholar
51. 1. Ramløv H
    2. Westh P

    (2001) Cryptobiosis in the eutardigrade Adorybiotus (Richtersius) coronifer: tolerance to alcohols, temperature and de novo protein synthesis

    Zoologischer Anzeiger 240:517–523.

    https://doi.org/10.1078/0044-5231-00062

    • Google Scholar
52. 1. Rebecchi L
    2. Altiero T
    3. Cesari M
    4. Bertolani R
    5. Rizzo AM
    6. Corsetto PA
    7. Guidetti R

    (2011) Resistance of the anhydrobiotic eutardigrade Paramacrobiotus richtersi to space flight (LIFE-TARSE mission on FOTON-M3)

    Journal of Zoological Systematics and Evolutionary Research 49:98–103.

    https://doi.org/10.1111/j.1439-0469.2010.00606.x

    • Google Scholar
53. 1. Schultz J
    2. Milpetz F
    3. Bork P
    4. Ponting CP

    (1998) SMART, a simple modular architecture research tool: identification of signaling domains

    Proceedings of the National Academy of Sciences of the United States of America 95:5857–5864.

    https://doi.org/10.1073/pnas.95.11.5857

    • Google Scholar
54. 1. Schwarz G

    (1978) Estimating the dimension of a model

    The Annals of Statistics 6:461–464.

    https://doi.org/10.1214/aos/1176344136

    • Google Scholar
55. 1. Simpson CL
    2. Patel DM
    3. Green KJ

    (2011) Deconstructing the skin: cytoarchitectural determinants of epidermal morphogenesis

    Nature Reviews Molecular Cell Biology 12:565–580.

    https://doi.org/10.1038/nrm3175

    • Google Scholar
56. 1. Stamatakis A

    (2014) RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies

    Bioinformatics 30:1312–1313.

    https://doi.org/10.1093/bioinformatics/btu033

    • Google Scholar
57. 1. Sugiura N

    (1978) Further analysis of the data by Akaike's information criterion and the finite corrections

    Communications in Statistics - Theory and Methods 7:13–26.

    https://doi.org/10.1080/03610927808827599

    • Google Scholar
58. 1. Tanaka S
    2. Tanaka J
    3. Miwa Y
    4. Horikawa DD
    5. Katayama T
    6. Arakawa K
    7. Toyoda A
    8. Kubo T
    9. Kunieda T

    (2015) Novel mitochondria-targeted heat-soluble proteins identified in the anhydrobiotic tardigrade improve osmotic tolerance of human cells

    PLoS ONE 10:e0118272.

    https://doi.org/10.1371/journal.pone.0118272

    • Google Scholar
59. 1. Wright JC

    (2001) Cryptobiosis 300 years on from van Leuwenhoek: what have we learned about tardigrades?

    Zoologischer Anzeiger 240:563–582.

    https://doi.org/10.1078/0044-5231-00068

    • Google Scholar
60. 1. Yamaguchi A
    2. Tanaka S
    3. Yamaguchi S
    4. Kuwahara H
    5. Takamura C
    6. Imajoh-Ohmi S
    7. Horikawa DD
    8. Toyoda A
    9. Katayama T
    10. Arakawa K
    11. Fujiyama A
    12. Kubo T
    13. Kunieda T

    (2012) Two novel heat-soluble protein families abundantly expressed in an anhydrobiotic tardigrade

    PLoS ONE 7:e44209.

    https://doi.org/10.1371/journal.pone.0044209

    • Google Scholar

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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