Trifluoroethanol-dependent reversibly condensing proteins (T-DRYPs) are identified from Ramazzottius varieornatus

We designed the experimental scheme shown in Fig 1A to identify tardigrade proteins that form condensates in response to dehydration-like stress in a reversible manner. We began with the lysate of the desiccation-tolerant tardigrade species R. varieornatus, because this species constitutively expresses the tolerance proteins and its genome sequence is available [16]. First, we added a desolvating agent, TFE to a soluble fraction of R. varieornatus lysate to induce condensation in a dehydration-like state. TFE is a cosolvent that affects the protein conformation by displacing water molecules from the surface of polypeptides [27] and/or destabilizing an aqueous solvation of polypeptide backbone [28], which indirectly promotes intramolecular hydrogen bonding and stabilizes the secondary structures of proteins. TFE is also known to promote alpha-helix formation in several desiccation-tolerance proteins, such as LEA and CAHS proteins as dehydration do [15,29,30]. The TFE-condensed proteins were collected as precipitates and resolubilized with TFE-free PBS to mimic rehydration (resolvation). Treatment with higher concentration of TFE increased the number of proteins detected in the resolubilized fraction (Figs 1B and S1). As treatment with 20% and 30% TFE had similar effects, we considered 20% TFE to be an adequate stress condition for this screening (S1 Fig). When treated with TFE at 20% or higher, many proteins, especially those with a high molecular weight, were detected in the irreversibly precipitated fraction, indicating that only the selected proteins were retrieved in the resolubilized fraction.

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Fig 1. Isolation and characterization of T-DRYPs.

(A) Experimental scheme of T-DRYP isolation from tardigrade lysate. (B) SDS-PAGE image of the resolubilized fractions with 0%, 10%, or 20% TFE treatment. (C) Comparison of the unstructured score distributions between all tardigrade proteins and T-DRYPs. (D) Enrichment analysis of the PANTHER protein class in T-DRYPs. Ribosomal proteins and cytoskeletal proteins were significantly enriched. The numbers of the corresponding proteins detected in T-DRYPs and all tardigrade proteomes are shown on the right, respectively. (E) Enrichment analysis of stress-related proteins in T-DRYPs. CAHS proteins were significantly enriched in T-DRYPs. (F) Venn diagram of T-DRYPs classified by up- or down-regulation upon desiccation in orthologs of 2 other tardigrade species. (G) Comparison of unstructured score distributions among the differently regulated protein groups in T-DRYPs. “Up-regulated” and “down-regulated” indicate up-regulated or down-regulated proteins in both species, respectively. Proteins up-regulated upon desiccation exhibited higher unstructured scores. Red and 2 black horizontal bars in violin plot indicate the 50th, 25th, and 75th percentiles, respectively. Statistical analyses were performed with the Wilcoxon rank sum test in (C) and the Steel–Dwass test in (G). The underlying numerical data are available in S4 Data (C, D, and G) in S2 Data (E) and in S1 Data (F). CAHS, cytoplasmic-abundant heat-soluble; T-DRYP, TFE-dependent reversibly condensing protein; TFE, trifluoroethanol.

https://doi.org/10.1371/journal.pbio.3001780.g001

We identified 336 proteins in the resolubilized fraction (20% TFE) by nanoflow liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) and collectively termed these proteins “TFE-Dependent ReversiblY condensing Proteins (T-DRYPs)” (S1 Data). Because reversible condensation is a characteristic property expected for unstructured proteins, we calculated the unstructured score of each protein in T-DRYPs by IUPred2A and compared the score distribution with those of all tardigrade proteins. As expected, unstructured proteins were significantly enriched in T-DRYPs (p < 2.2e-16, Wilcoxon rank sum test; Fig 1C). We assigned Drosophila melanogaster orthologs for tardigrade proteins and performed enrichment analysis of PANTHER Protein class or Gene Ontology term in T-DRYPs. The results revealed that ribosomal proteins and actin-related cytoskeletal proteins were well concentrated in T-DRYPs (Figs 1D and S2). Among T-DRYPs, however, 105 (31%) proteins had no apparent fly orthologs and T-DRYPs contain many tardigrade-unique proteins (21%) including known tolerance proteins like CAHS proteins (S1 Data). Therefore, we expanded the enrichment analyses to the previously annotated tardigrade tolerance protein families that contain more than 5 members [16] and revealed the significant enrichment of CAHS, LEA, HSP20, HSP70, and peroxiredoxin families in T-DRYPs (p < 0.01, chi-square test; Fig 1E and S2 Data), suggesting that our new screening scheme concentrates desiccation-tolerance related proteins to the resolubilized fraction. To evaluate this possibility further, we classified T-DRYPs into 3 groups: stress-up-regulated groups, stress-down-regulated groups, and the others. R. varieornatus is one of the toughest tardigrade species that constitutively expresses stress-related genes [16]. Thus, we utilized gene expression data of 2 closely related tardigrades, Hypsibius exemplaris and Paramacrobiotus metropolitanus (formerly Paramacrobiotus sp. TYO), both of which exhibit strong up-regulation of tolerance gene expression upon desiccation [11,17,31]. Of 336 T-DRYPs, 315 proteins had orthologs in both species and 72 genes were up-regulated during dehydration (Fig 1F and S1 Data). Statistical analysis indicated that the up-regulated proteins were significantly enriched in T-DRYPs compared with the tardigrade proteome (p = 9.53e-29, chi-square test; S2 Data). In addition, the up-regulated proteins also exhibited a much higher unstructured score (Fig 1G), suggesting that tolerance-related unstructured proteins were well concentrated in the resolubilized fraction in our scheme. Because CAHS proteins were highly enriched in the T-DRYPs (Fig 1E), and also 3 major bands in the resolubilized fraction were separately identified as CAHS12, CAHS3, and CAHS8 (Figs 1B and S3), we focused on these 3 CAHS proteins for further analyses.

CAHS3, CAHS8, and CAHS12 reversibly assemble into filaments or granules in animal cells depending on hyperosmotic stress

To visualize the stress-dependent condensation, 3 CAHS proteins, such as CAHS3, CAHS8, and CAHS12 proteins were separately expressed as a GFP-fused protein in human cultured HEp-2 cells and the distribution changes of these fusion proteins were examined upon exposure to a hyperosmotic stress, which induces water efflux like dehydration stress [32]. In an unstressed condition, CAHS3-GFP broadly distributed in the cytosol, whereas CAHS8-GFP and CAHS12-GFP distributed broadly in both the cytosol and the nucleus with CAHS12-GFP showing a slight preference for the nucleus (Fig 2A). When exposed to hyperosmotic medium supplemented with 0.4 M trehalose, CAHS3-GFP condensed and formed a filamentous network in the cytosol (Fig 2A and 2B). Similar filament formation was observed when CAHS3 alone was expressed without GFP (S4 Fig), suggesting that filament formation is an intrinsic feature of CAHS3 protein rather than artifact of fusion with GFP. CAHS12-GFP also formed filaments in the cytosol and more prominently in the nucleus in a majority of cells, though granule-like condensates were also observed in the nucleus of approximately 34% of the cells (Figs 2B and S5). CAHS8-GFP predominantly formed granule-like condensates especially in the nucleus, but filaments were also observed in the cytosol in a small population (approximately 3%) of the cells. Similar distribution changes were observed even when GFP was fused to the opposite site in CAHS proteins (S6 Fig), while GFP alone did not exhibit such drastic changes. When hyperosmotic stress was removed by replacing with isosmotic medium, all CAHS condensates, both filaments and granules, rapidly dispersed (Fig 2A and 2B). Hyperosmotic stress by other supplemented osmolytes, such as 0.2 M NaCl or 0.4 M sorbitol, which have an equivalent osmolarity to 0.4 M trehalose, induces similar filament or granule formation, suggesting that the hyperosmotic stress itself is the driver of condensation rather than specific effects of each osmolyte (S7 Fig). Similar reversible condensations of CAHS proteins were also observed when expressed in Drosophila cultured S2 cells (S8 Fig and S1 Movie), indicating that the stress-dependent filament/granule condensations are intrinsic features of CAHS proteins commonly observed in animal cells of taxonomically distant species.

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Fig 2. Reversible formation of filaments or granules by CAHS3, CAHS8, and CAHS12 proteins in response to a hyperosmotic stress.

(A) Distribution changes in AcGFP1-tagged CAHS3, CAHS8, or CAHS12 proteins in HEp-2 cells during the transient hyperosmotic treatment with HBSS containing 0.4 M trehalose. Blue indicates Hoechst33342 staining of nuclei. (B) The proportion of distribution patterns (filaments, granules, or dispersed) of each CAHS protein in human cells. (C and D) FRAP analyses of CAHS3-GFP in human cells in dispersed state under an isosmotic condition (C, n = 7) and in a filament-formed state under a hyperosmotic condition (D, n = 6). (E) Confocal images of AcGFP1-tagged CAHS3 proteins and fluorescently labeled cytoskeletal proteins in HEp-2 cells under a hyperosmotic condition. White arrowheads indicate slight co-localization of CAHS3 proteins and actin filaments. (F and G) Time-lapse images of filament formation or deformation of CAHS3-GFP in human cells (see also S2 and S3 Movies). CAHS3-GFP first condensed into granules (155 s) and then elongated into filaments (355 s) as indicated by white arrowheads (F). CAHS3-GFP filaments simultaneously collapsed and dispersed (398 s) (G). Time since the medium exchange to hyperosmotic (F) or isosmotic (G) solution is shown in each image. (H) Fluorescent images of HEp-2 cells co-expressing pairs of CAHS3, CAHS8, and CAHS12 proteins with a different fluorescent-tag under a hyperosmotic condition. CAHS3 co-localized with neither CAHS8 nor CAHS12. In contrast, CAHS8 well co-localized with CAHS12 filaments. White arrowheads indicate representative co-localization. Scale bar, 10 μm in (A, E, and H), 2 μm in (F and G). The underlying numerical data are available in S4 Data (B–D). CAHS, cytoplasmic-abundant heat-soluble; FRAP, fluorescence recovery after photobleaching; HBSS, Hanks’ Balanced Salt Solution.

https://doi.org/10.1371/journal.pbio.3001780.g002

Granule-like condensates of CAHS8 resemble droplet structures formed by intrinsically disordered proteins via liquid–liquid phase separation. To test this possibility, we examined the effect of 1,6-hexanediol, a disruption reagent of liquid-like condensates. After treatment with 5% 1,6-hexanediol for 30 min, the well-known droplet-forming protein FUS effectively dispersed, while several CAHS8 granules in the nucleus also dispersed but much less effectively than FUS protein granules (S9 Fig), suggesting that CAHS8 granules were partly liquid like. In contrast, the filament structures of CAHS3 or CAHS12 were not affected by the hexanediol treatment, suggesting that CAHS3 and CAHS12 filaments were in a static solid-like state. To further assess the staticity of CAHS filaments, we performed fluorescence recovery after photobleaching (FRAP) analysis on CAHS3-GFP both before and after exposure to hyperosmotic stress. In unstressed cultured cells, CAHS3-GFP was broadly distributed in the cytosol and the bleached fluorescence was rapidly recovered (Fig 2C), indicating their high mobility nature. In contrast, under hyperosmotic stress, CAHS3-GFP filaments exhibited almost no fluorescence recovery after bleaching (Fig 2D), suggesting that CAHS3 proteins formed static filaments in response to a hyperosmotic stress. The filamentous networks formed by CAHS proteins resembled cytoskeletal structure. To examine possible cooperation between CAHS filament formation and other cytoskeletal structures or organelles, we performed co-localization analyses and observed no co-localization between filament-forming CAHS proteins and any examined intracellular structures except for slight co-localization with actin filaments (Figs 2E and S10A–S10C). GFP alone also exhibited slight co-localization with actin filaments and actin polymerization inhibitor did not disrupt filament formation of CAHS3 and CAHS12 proteins (S10D, S10E, and S11 Figs), suggesting that CAHS filament formation is also independent from actin filaments. These results suggested that CAHS molecules freely disperse in an unstressed condition, but upon the exposure to hyperosmotic stress, CAHS molecules are firmly integrated into an additional cytoskeleton-like filaments.

To elucidate the process of filament formation and deformation in more detail, we captured time-lapse images of cells expressing CAHS3-GFP while changing the stress conditions by high-speed super-resolution microscopy. Approximately 2.5 min after the medium was changed to a hyperosmotic condition by a perfusion device, CAHS3-GFP began to condense simultaneously at many sites in the cells and rapidly formed fibril structures. The fibrils then further extended in a few dozen seconds (Fig 2F and S2 Movie). When the hyperosmotic stress was removed by changing to an isosmotic medium, CAHS3 filaments simultaneously began to loosen and gradually dispersed in approximately 6 min (Fig 2G and S3 Movie). The initial condensation of CAHS3 and the granule formation of CAHS8 likely occurred via phase separation, which frequently leads to co-condensation of multiple proteins, especially those containing similar motifs [33]. CAHS proteins share several conserved motifs and could thus cooperatively form the same condensates. To examine this, we co-expressed pairs of the 3 CAHS proteins labeled with different fluorescent proteins in human cells. Under hyperosmotic stress, CAHS3 filaments did not co-localize with CAHS8 granules or CAHS12 filaments (Fig 2H). In contrast, CAHS8 largely co-localized with CAHS12 filaments throughout the cell, suggesting that the granule-forming CAHS8 cooperatively forms the filament structure with other CAHS proteins such as CAHS12.

Secondary structure in the conserved C-terminal region is responsible for CAHS filament formation

To reveal the structural basis of CAHS filament formation, we first performed de novo motif search and found 10 conserved motifs by comparing 40 CAHS proteins of 3 tardigrade species, R. varieornatus, H. exemplaris, and P. metropolitanus (S12 and S13 Figs and S3 Data). In particular, we found that 2 C-terminal motifs (CR1 and CR2) are highly conserved in all CAHS family members except 1 CAHS protein of H. exemplaris (S12 Fig). To determine the region responsible for filament formation, we generated a series of truncated mutant proteins of CAHS3 or CAHS12 either N-terminally or C-terminally, and examined their filament formation in human cultured cells under a hyperosmotic stress (Figs 3A, 3B, and S14). In CAHS3, N-terminal deletion to motif 3 or C-terminal deletion to CR2 drastically impaired filament formation and instead granule formation was frequently observed in the cytosol (Figs 3B, S15A and S15B). Accordingly, we designed a truncated mutant consisting of the minimum required region from motif 3 to CR2 (motif 3-motif H1-CR1-CR2) and revealed that this region is sufficient for the filament formation by CAHS3 protein (Figs 3B and S15C). Similarly, in CAHS12 protein, the region consisting of CR1, CR2, and the 2 preceding motifs (motif H2-motif H3-CR1-CR2) was shown to be necessary and sufficient for the filament formation (S14 Fig). These results indicated that 2 highly conserved motifs (CR1 and CR2) and 2 preceding motifs (65 to 85 residues) play an essential role in the filament formation of both CAHS3 and CAHS12 proteins.

In these identified regions responsible for the filament formation, extensive helix and coiled-coil structures were predicted by the secondary structure prediction tool, JPred4 and COILS (Figs 3C and S16). The coiled-coil structure is the key structural basis for the polymerization of IFs [34]. To test the contribution of these predicted secondary structures in filament formation, we generated 2 mutants for each CAHS3 and CAHS12 by substituting leucine with proline, which are predicted to disrupt the helical and coiled-coil structures of CR1 or CR2, respectively (Figs 3C and S17) [35]. As expected, all coiled-coil disruption mutants exhibited significantly impaired filament formation and instead formed granules (Figs 3C and S17–S19). The double mutation (CAHS3-L207P-L236P) further suppressed filaments formation and even reduced granule formation (Figs 3C and S20). These results suggested that the secondary structures of both CR1 and CR2 are an important basis for the filament formation of CAHS3 and CAHS12 proteins.

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Fig 3. Secondary structure in the conserved C-terminal region is responsible for the CAHS filament formation and gelation.

(A) Schematic diagrams of CAHS3 proteins. “CR1” and “CR2” indicate putative helical motifs highly conserved among almost all CAHS family members. “H1” indicate putative helical conserved motifs; “1,” “2,” and “3” indicate other conserved motifs. (B) Schematic diagrams and the corresponding distribution patterns of the truncated mutants of CAHS3. Quantified cell proportions of the distribution patterns under a hyperosmotic condition are shown as a stacked bar graph. Confocal images are shown for the representative distribution pattern of the corresponding CAHS mutants. Blue indicates Hoechst33342 staining of nuclei. (C) Effects of a helix-disrupting mutation by substituting leucine with proline on filament formation of CAHS3. Schematic structure and the coiled-coil score predicted by COILS are shown for both wild-type and proline substitution mutants. Asterisks indicate the sites of proline substitutions. Substitution with proline substantially decreased the coiled-coil score in the corresponding region. Confocal images show representative distribution patterns of the corresponding CAHS proteins. Enlarged image is shown as superimposition in each panel. Blue indicates Hoechst33342 staining of nuclei. Quantified cell proportions of each distribution pattern are shown as stacked bar plots on the right. (D) In vitro time-lapse confocal images of fibril formation of CAHS3-GFP proteins (1.24 mg/mL) after adding TFE (final 20%). GFP is a non-filament forming control. (E) TFE-dependent reversible gel-formation of CAHS3 proteins. By adding TFE (final 20%), CAHS3 protein solutions (4.0 mg/mL) became turbid and transited into a gel-like state. The gels spontaneously liquefied within several minutes (shown in white letters) after exposure to air. (F) Filament-impaired CAHS3-L207P mutant protein solutions failed to transit into a gel-like state under 20% TFE. (G) Minimum filament-forming CAHS3 truncated protein (CAHS3-min) solution reversibly solidified under 20% TFE like full-length CAHS3 protein. Scale bar, 10 μm in (B and C), 2.5 μm in superimposition in (C), 20 μm in (D), 2 mm in (E–G). The underlying numerical data are available in S4 Data (B and C). CAHS, cytoplasmic-abundant heat-soluble; TFE, trifluoroethanol.

https://doi.org/10.1371/journal.pbio.3001780.g003

In vitro reversible gel transition of CAHS proteins depending on desolvating agent and salt

To examine whether CAHS proteins alone are sufficient to form filaments, we performed in vitro experiments using purified CAHS3-GFP proteins. Under an unstressed condition, the uniform distribution of CAHS3-GFP proteins was observed under a confocal microscope (Fig 3D). When the desolvating agent TFE was added to induce a dehydration-like conformational change as in our initial screening, CAHS3-GFP immediately condensed and formed mesh-like fibril networks after 1 min. This result indicated that CAHS3 proteins alone can sense the changes in the condition and form filaments without the assistance of other proteins.

When TFE was added to the solution containing a higher concentration of purified CAHS3 protein (final 4 mg/mL; S21 Fig), the protein solution immediately became turbid, and the solution was solidified into a gel-like state (Fig 3E). When the CAHS3 gel in the tube was spread onto parafilm, the CAHS3 gel spontaneously liquefied within approximately 10 min. We speculated that volatilization of TFE relieved the desolvating stress, thereby making the CAHS3 gel resoluble. Consistently, washing with TFE-free PBS also redissolved the gelated CAHS3 (S22A Fig). While the control protein BSA was not solidified in the same condition (S22B Fig), CAHS8 and CAHS12 exhibited a similar TFE-dependent reversible gel-transition like CAHS3, but the gel of CAHS8 was much smaller than those of other CAHS proteins (S22C and S22D Fig), suggesting differences in the propensity for gelation among CAHS proteins. We also examined whether other stressors that could emerge during dehydration induce CAHS gelation and revealed that an increased concentration of salt (2 M NaCl) also induced the gel transition of CAHS3 proteins, while a molecular crowding agent (20% polyethylene glycol) caused turbidity, but no gelation (S23 Fig). The salt-induced gel persisted even after exposure to air on parafilm, possibly because salt cannot evaporate (S23A Fig). The granule-forming CAHS8 only formed a very small gel in vitro, implying a possible relationship between the filament-forming ability in cells and the gel-forming ability in vitro. This notion was supported by the fact that the filament-impaired CAHS3-L207P mutant protein failed to form the gel in vitro (Fig 3F). In contrast, minimum CAHS3 protein possessing the filament-forming ability (CAHS3-min) successfully formed the gel in vitro upon the addition of TFE and this transition was reversible as in full-length CAHS3 (Fig 3G), suggesting that the filament-forming ability underlies the gel transition of CAHS proteins in vitro.

CAHS confers the mechanical resistance against deformation forces on cell-like microdroplets and insect cells

To reveal what the gelation of CAHS proteins provides, we evaluated the effects of CAHS gelation on the mechanical properties of cells using cell-like microdroplets covered with a lipid layer. The elasticity of the microdroplets was examined by measuring the elongation length in a micropipette while aspirating with a certain pressure. Microdroplets containing uniformly distributed CAHS3-GFP exhibited continuous elongation exceeding 50 μm under very small pressure (<<0.5 kPa), indicating that they were not elastic and in a liquid phase (Fig 4A–4C). On the other hand, the addition of salt induced the filament formation of CAHS3-GFP and the corresponding microdroplets exhibited significant elasticity (Young’s modulus approximately 2.0 kPa in average), indicating that the CAHS3-GFP droplets gelated and then physically hardened. Microdroplets containing GFP alone were not elastic regardless of the addition of salt (Fig 4B and 4C).

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Fig 4. CAHS confers the mechanical resistance against deformation forces on cell-like microdroplets and insect cells.

(A) Representative fluorescent images of a microdroplet containing CAHS3-GFP in the absence or presence of additional NaCl. Scale bar, 5 μm. (B) Representative response curves of the elongation length of microdroplets containing CAHS3-GFP or GFP alone under a very small pressure (<<0.5 kPa). Continuous elongation exceeding 50 μm indicates not elastic and in a liquid phase. (C) Comparison of the elasticity (Young’s modulus) among droplets containing CAHS3-GFP or GFP with or without NaCl addition. Data are presented as average ± SE. (D) The effects of CAHS3-expression on the cortical elasticity of Drosophila S2 cells under hyperosmosis. CAHS3-stably expressing cells exhibited higher elasticity compared to the control cells transfected with empty vector under a hyperosmotic condition supplemented with 0.4 M trehalose for 3 h. Gray and red dots indicate the values of each measurement. Black dots and bars indicate averages and standard errors, respectively. (E) Comparison of cell volume changes by hyperosmotic stress between CAHS3-expressing cells and control cells. The relative cell volume was calculated by dividing the volume under hyperosmotic stress by the averaged cell volume under isosmotic conditions. Center bar and edges indicate 50th, 25th, and 75th percentiles, respectively, and whiskers correspond to the 1.5 interquartile range. (F and G) Comparison of cell viability between CAHS3-expressing cells and control cells under an isosmotic condition (F) and a hyperosmotic condition for 48 h (G). PI was used to determine dead cells. Survival rates were examined in 6 wells for each condition by counting >500 cells/well. Statistical analyses were performed with the Wilcoxon rank sum test in (D) and (E), and Student t test in (F) and (G); n.s. means not significant in the statistical tests. The underlying numerical data are available in S4 Data (B–G). CAHS, cytoplasmic-abundant heat-soluble; PI, propidium iodide.

https://doi.org/10.1371/journal.pbio.3001780.g004

To further determine whether CAHS proteins also stiffen animal cells, we established a Drosophila S2 cell line stably expressing CAHS3. S2 cells lack canonical cytoplasmic IFs as tardigrade cells do [24] and thus it would be suitable to measure the effect of CAHS filamentation. Measuring the cell stiffness by atomic force microscope (AFM) revealed that under an unstressed condition, the CAHS3-expressing cells exhibited no significant difference in the elasticity with that of the control cells transfected with empty vector. Under a hyperosmotic condition, control cells exhibited higher elasticity than that in an unstressed condition, but the CAHS3-expressing cells exhibited significantly further higher elasticity than that of the control cells under the same condition (p < 0.05; Fig 4D), which is consistent with the results using microdroplets. Hyperosmotic stress reduces the cell volume through osmotic pressures [32]. As CAHS3-expressing cells exhibited higher elasticity under a hyperosmotic condition, they somewhat counteract the osmotic pressure and might resist the cell shrinkage. To examine this possibility, we measured the cell volume changes by exposure to hyperosmotic stress. As shown in Fig 4E, CAHS3-expressing cells retained the cell volume significantly better than the control cells (p < 0.001; Fig 4E). These results suggest that under a filament-forming condition, CAHS3 proteins stiffen cells and protect them from deformation stress caused by water-efflux stress. Furthermore, we also examined the effect of CAHS3 on cell viability after exposure to hyperosmotic stress. Cell viability was evaluated by the exclusion of propidium iodide (PI), which is an indicator of cell integrity. Under an unstressed condition, CAHS3 expression did not affect the cell viability, but after 48 h treatment with hyperosmotic stress, CAHS3-expressing cells exhibited the increased cell viability (Fig 4F and 4G). The cell stiffening by CAHS proteins may contribute to the stabilization of cell structure and the survival of cells during the dehydration-like process.

Animals

We used the previously established YOKOZUNA-1 strain of the desiccation-tolerant tardigrade R. varieornatus reared on water-layered agar plate by feeding alga Chlorella vulgaris (Recenttec K. K., Japan) at 22°C as described previously [36].

Identification of trifluoroethanol-dependent reversibly condensing proteins

Prior to protein extraction, tardigrades were starved for 1 day to eliminate digestive food. Approximately 400 R. varieornatus were collected and extensively washed with sterilized Milli-Q water to remove contaminants. Tardigrades were rinsed with lysis buffer, phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.76 mM KH₂PO₄ (pH 7.4)) containing complete protease inhibitors (Roche) and transferred to a 1.7 mL tube. Tardigrades were homogenized in 20 μL lysis buffer using a plastic pestle on ice. The pestle was rinsed with an additional 20 μL of lysis buffer collected in the same tube. After centrifugation at 16,000 × g for 20 min at 4°C, the supernatant was recovered as a soluble protein extract. To mimic dehydration stress, the desolvating agent, TFE, was added (final concentration 10%, 20%, or 30%), and the mixture was incubated on ice for 1 h to allow complete induction of condensation. After centrifugation at 16,000 × g for 20 min, the supernatant was removed as a TFE-soluble fraction and the remaining precipitate was washed twice by lysis buffer containing TFE at the same concentration. The washed precipitate was resuspended in lysis buffer without TFE and incubated at room temperature for 30 min to facilitate resolubilization. After centrifugation at 16,000 × g at 4°C, the supernatant was recovered as a resolubilized fraction. The fractions were analyzed by SDS-PAGE and proteins were visualized using a Silver Stain MS Kit (Fujifilm). Three selected bands were excised and separately subjected to mass spectrometry. Comprehensive identification of T-DRYPs was achieved by shot-gun proteomics of the resolubilized fraction. Briefly, proteins in gel slices or in the fraction were digested with trypsin and fragmented peptides were analyzed by nanoLC-MS/MS. Proteins were identified using MASCOT software (Matrix Science). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the jPOST repository with the dataset identifier PXD030241 and can be retrieved at http://proteomecentral.proteomexchange.org/cgi/GetDataset?ID=PXD030241.

In silico structure predictions

The unstructured score of the proteins was calculated by IUPred2A [48]. IUPred2A produces the score for each amino acid position in a protein, and an average value was used as a score for each protein. A de novo protein sequence motif search in CAHS protein families was performed by the motif discovery tool, MEME version 5.0.4 [49] (https://meme-suite.org/meme/tools/meme). The parameters were as follows: (occurrence per sequence = 0 or 1; the maximum number to be found = 10; the motif width = 6 to 50). The secondary structures of CAHS3, CAHS8, and CAHS12 proteins were predicted by Jpred4 [50] (https://www.compbio.dundee.ac.uk/jpred/). The coiled-coil regions of CAHS3 and CAHS12 were predicted by COILS [51] (https://embnet.vital-it.ch/software/COILS_form.html or locally executing the software available at ftp://ftp.ebi.ac.uk/pub/software/unix/coils-2.2/). The 3D structure prediction of the CAHS-min protein homo-dimer was performed by Alphafold2 [41] (https://colab.research.google.com/github/sokrypton/ColabFold/blob/main/AlphaFold2_complexes.ipynb). The 3D structures were visualized with UCSF ChimeraX v.1.2 [52].

Enrichment analysis

To utilize well-annotated information in the model organism D. melanogaster, we assigned a D. melanogaster ortholog for each R. verieornatus protein by a reciprocal BLAST search. We assigned 231 fly orthologs for 336 T-DRYPs and 7,361 fly orthologs for all 19,521 R. varieornatus proteins. Using the assigned fly orthologs, we performed enrichment analyses with PANTHER Overrepresentation Test [53] (PANTHER Protein Class version 16.0, Fisher’s test; http://pantherdb.org/) and Metascape [54] (GO Cellular Components; https://metascape.org/). The list of fly orthologs for all R. varieornatus proteins was used as a reference in the enrichment analyses.

Among tardigrade stress-related proteins described previously [16], 7 protein families containing more than 5 members were selected for the enrichment analysis (S2 Data). Enrichment of each family in T-DRYPs was statistically examined by Fisher’s exact test using R. Enrichment of up-regulated genes was similarly examined except using a chi-square test.

Differential gene expression analysis

Transcriptome data at a hydrated state and a dehydrated state were retrieved from the public database (DRR144971-DRR144973 and DRR144978-DRR144980 for Paramacrobiotus metropolitanus; SRR5218239-SRR5218241 and SRR5218242-SRR5218244 for Hypsibius exemplaris, respectively). The genome sequence of P. metropolitanus was retrieved from the public database under accession numbers BHEN01000001-BHEN01000684 [11]. The genome sequence of H. exemplaris v3.0 was retrieved from http://www.tardigrades.org. RNA-seq reads were mapped to the genome sequence using HISAT2 v.2.1.0 [55]. Read counts for each gene region were quantified by featureCounts in SubRead package v.1.6.3 [56] and statistically compared by R package DESeq2 [57]. The genes with FDR < 0.01 were considered as differentially expressed genes. Orthologous gene relationships were determined by reciprocal BLAST searches among 3 tardigrade species.

Cell lines

We obtained Hep-2 cells (RCB1889) from RIKEN BioResource Center (BRC). The identity of the cell line was validated by short tandem repeat profiling and the cell line was negative for mycoplasma contamination (RIKEN BRC). The cell was maintained in minimum essential medium (Nacalai Tesque) containing 10% fetal bovine serum (FBS, Cosmo Bio or BioWest) at 37°C, 5% CO₂. Drosophila S2 cells (Gibco) were cultured at 28°C in Schneider’s Drosophila Medium (Gibco) supplemented with 10% heat-inactivated FBS (BioWest) and penicillin-streptomycin mixed solution (Nacalai Tesque).

Plasmids

CAHS3, CAHS8, and CAHS12 coding sequences were amplified from the corresponding EST clones of R. varieornatus [16] and inserted into pAcGFP1-N1 or pAcGFP1-C1 (Clontech) with (GGGGS)₃ linker using In-Fusion HD Cloning Kit (Takara). Plasmids to express CAHS deletion mutants (CAHS3Δctail, CAHS3ΔCR2-C, CAHS3ΔN-M2, CAHS3ΔN-M3, CAHS3-min, CAHS12Δctail, CAHS12ΔCR2-C, CAHS12ΔN-M1, CAHS12ΔN-H2, and CAHS12-min) or leucine-to-proline substitution mutants (CAHS3-L207P, CAHS3-L236P, CAHS3-L207P-L236P, CAHS12-L204P, and CAHS12-L241P) were generated by inverse PCR and ligation or PCR-based site directed mutagenesis. The CAHS3/8/12-mScarlet-I expression vector was generated from CAHS3/8/12-GFP expression vector by replacing AcGFP1 coding sequences with mScarlet-I sequence fragments [58] synthesized artificially (IDT). Expression constructs for various cytoskeleton or organelle marker proteins were obtained from Addgene (S1 Table). For bacterial expression of His₆-tagged CAHS proteins, CAHS3, CAHS8, or CAHS12 coding sequences were amplified and inserted into pEThT vectors [15], and CAHS3-GFP was similarly inserted into a pCold-I vector (Takara). For expression in Drosophila cells, codon-optimized CAHS3, CAHS8, CAHS12, and AcGFP1 DNA fragments were synthesized (Gene Universal) and inserted into pAc5.1/V5-His A vector (Invitrogen). The FUS-Venus plasmid was a kind gift from Dr. Tetsuro Hirose.

Live cell imaging under hyperosmosis

We used Hep-2 cells for live-imaging of fluorescently labeled proteins because Hep-2 cell were well sticky even under a stress condition and enabled precise inspections. Hep-2 cells were transiently transfected with an expression vector of fluorescently labeled proteins using Lipofectamine LTX & Plus Reagent (Invitrogen) for 48 h before stress exposure. Prior to microscopy, the medium was replaced with Hanks’ Balanced Salt Solution (HBSS) without the dications and phenol red. For exposure to hyperosmotic stress, the buffer was replaced with HBSS containing 0.4 M trehalose. The cells were stained with Hoechst 33342 (5 μg/mL, Lonza) to visualize nuclear DNA. Fluorescent signals were observed using a confocal microscope LSM710 (Carl Zeiss). The number of cells for each CAHS distribution pattern, such as dispersed, granules, or filaments, were counted by 2 independent investigators and averaged counts were used. For time-lapse imaging in 3D space, we used the LSM-980 with Airyscan to perform super-resolution imaging. From the z-stack images, we generated orthogonal projections using ZEN 2.6 software. In time-lapse imaging experiments, a perfusion system KSX-Type1 (Tokai Hit) was used to replace the buffer. To visualize actin filaments by chemical staining, Hep-2 cells were treated with silicon-rhodamine dye probing actin (SiR-actin, Spirochrome) in HBSS containing the drug efflux inhibitor verapamil (10 μM, Tokyo Chemical Industry) for 2 h. For actin polymerization inhibition experiments, cells were treated with cytochalasin B (5 μM, Nacalai Tesque) for 60 min. Cells were then observed by a confocal microscope LSM-710 (Carl Zeiss).

Fluorescence recovery after photobleaching (FRAP) analysis

Hep-2 cells were transiently transfected with the expression construct of CAHS3-GFP. The transfected cells were then exposed to isosmotic HBSS or hyperosmotic buffer, HBSS containing 0.4 M trehalose, to analyze the mobility of CAHS3-GFP in the dispersed or filament state, respectively. FRAP experiments were performed at room temperature using a confocal fluorescence microscope (FV1200, Olympus). A spot approximately 0.77 μm in diameter was photobleached at 100% laser power (wavelength 473 nm), and the fluorescence recovery curves were analyzed using the Diffusion Measurement Package software (Olympus). The fluorescence intensity was normalized by the initial intensity before photobleaching.

Sensitivity to 1,6-hexanediol treatment

Hep-2 cells were transfected with expression vectors of CAHS3/8/12-AcGFP1 or FUS-Venus. After 48 h, cells were exposed on minimum essential medium supplemented with 0.4 M trehalose and 10% FBS for 1 h to induce the formation of granules or filaments. FUS protein was used as a control as it is known to be incorporated into liquid droplets under hyperosmosis [59]. After the addition of a liquid droplet disruptor, 1,6-hexanediol (final 5%), fluorescent images were captured at 0 and 30 min later by a confocal microscope LSM710 (Carl Zeiss). The fluorescence intensity was measured by Fiji and normalized to the initial fluorescence intensity of the granules or filaments.

Immunofluorescence

Hep-2 cells expressing CAHS3 or CAHS3 mutants were exposed to HBSS containing 0.4 M trehalose for 60 min to induce filament formation. The cells were then fixed in methanol at −30°C for 3 min and washed 3 times with PBS containing 0.1% Tween 20 (PBS-T). The cells were blocked with 2% normal goat serum (Abcam) for 1 h at room temperature and then reacted with 1/200 diluted antiserum against CAHS3 in 2% normal goat serum for 1 h at room temperature or 16 h at 4°C. The cells were washed 3 times with PBS-T and then reacted with 1/1,000 diluted Alexa Fluor546 goat anti-guinea pig secondary antibody (Invitrogen) and 1 μg/mL DAPI in 2% normal goat serum for 1 h at room temperature. Fluorescent signals were observed using a confocal microscope LSM710 (Carl Zeiss).

Protein preparation

Recombinant proteins were expressed as N-terminally His₆-tagged proteins in Escherichia coli BL21(DE3) strains. CAHS3, CAHS8, and CAHS12 proteins were expressed using pET system (Novagen) essentially as described previously [15]. CAHS3-GFP and AcGFP1 were expressed using a cold shock expression system (Takara) essentially as described previously [60]. Bacterial pellets were lysed in PBS containing complete EDTA-free protease inhibitors (Roche) by sonication. For CAHS3, CAHS8, and CAHS12, the supernatant was heated at 99°C for 15 min to retrieve heat-soluble CAHS proteins in a soluble fraction as described previously [15]. From the soluble fraction, His₆-tagged proteins were purified with Ni-NTA His-Bind Superflow (Novagen) and dialyzed against PBS using a Pur-A-Lyzer Midi Dialysis Kit (Merck).

In vitro polymerization of CAHS3-GFP proteins

Purified CAHS3-GFP or AcGFP1 protein solution in PBS (approximately 40 μM) was directly dropped on cover glass (MATSUNAMI), and fluorescent images were captured by a confocal microscope LSM710 (Carl Zeiss). To induce the polymerization of CAHS3, an equal amount of PBS containing TFE was added (final 20%), and time-lapse images were captured every 5 s.

In vitro gelation

Purified recombinant CAHS protein solution (5 mg/mL) was placed in a 0.2-mL tube. Inducing reagents such as TFE (final 20%), polyethylene glycol (final 20%), or NaCl (final 2 M) were added to the protein solution and incubated at room temperature for 10 min. Then, the tube contents were spread out on parafilm to check if it had solidified into a gel-like state or remained in a liquid state. Photos were obtained by a digital camera with a short focal length (Olympus TG-6).

Preparation of cell-like microdroplets

Cell-like microdroplets coated with a lipid layer of phosphoethanolamine (Nacalai Tesque) were prepared in an oil phase. First, dry films of the lipids were formed at the bottom of a glass tube. Mineral oil (Nacalai Tesque) was then added to the lipid films followed by 90 min of sonication. The final concentration of the lipid/oil solution was approximately 1 mM. Next, 10 vol % of the protein solution (40 μM GFP-labeled CAHS3 or 40 μM GFP) was added to the lipid/oil solution at approximately 25°C. After emulsification via pipetting, approximately 40 μL sample containing the microdroplets was placed on a glass-bottom dish. To condense the proteins inside the droplets upon dehydration, we added 40 μL salted oil. Mechanical measurements were performed 90 min after the droplet volume was approximately halved. For fluorescent imaging, 21 μM CAHS3-GFP and 171 μM CAHS3 were mixed and used.

Measurement of the elasticity of droplets by micropipette aspiration

The elasticity of the cell-like microdroplets was evaluated by a micropipette aspiration system as reported previously [61]. The surface elasticity (Young’s modulus), E, is derived from the linear relationship between the elongation length into the micropipette, ΔL, and the aspiration pressure, ΔP: E = (3ΔPRpΦ/2π)/ΔL, wherein Rp and Φ are the micropipette inner radius and wall function, which is derived from the shape of the micropipette. We used a micropipette with an Rp smaller than × 0.4 of the microdroplet radius R. The value of Φ is 2.0. An increase in ΔL to above 50 μm under a very small ΔP (<<0.5 kPa) indicates that the microdroplet is in liquid phase. In the case of the elastic gel phase, a linear relationship between ΔL and ΔP was confirmed for the small deformation within ΔL < 5 μm and ΔP < 3 kPa. Under these conditions, we derived the values of E. The temperature was approximately 25°C.

Establishment of stably transfected cell line of Drosophila S2 cells

The expression vector Ac5-STABLE2-neo was obtained from Addgene (#32426) [62], and then the coding sequence of FLAG-mCherry was replaced with the codon-optimized CAHS3 coding sequence (Gene Universal) to express CAHS3-T2A-EGFP-T2A-neoR under the control of Ac5 promoter. The empty vector was constructed by deleting FLAG-mCherry from Ac5-STABLE2-neo, which was designed to express T2A-EGFP-T2A-neoR driven by the same Ac5 promoter. Drosophila S2 cells were transfected using a cationic liposome reagent Hilymax (Dojindo) with the expression construct or the empty vector above. We established stably transfected cells by culturing for 6 weeks under the drug selection with G418 disulfate (2,000 μg/mL, Nacalai Tesque).

AFM measurement of elasticity of S2 cells expressing CAHS3 protein

Drosophila S2 cells stably transfected with the CAHS3 expression construct and empty vector were cultured on PLL-coated coverslips (MATSUNAMI) at least 1 day before the measurement for cell attachment. For hyperosmotic treatment, the culture medium was replaced with the one containing 0.4 M trehalose 1 h before the measurement. The force spectroscopy was conducted using NanoWizard 3 Ultra AFM (Bruker) with an inverted microscope Olympus IX70 at 22°C. Cantilevers BL-AC40TS (Olympus) with a nominal spring constant of 0.09 N/m was calibrated using the thermal noise method for each experiment. Photodetector sensitivity was determined by fitting a line to the slope of the force distance curve acquired on the glass substrate. Indentation tests were performed at the speed of 2 μm/s for both approach and retraction, and the tests were repeated 16 times for each single cell. Young’s moduli of the cells were obtained by fitting the Hertz model to the force curves using indentation depths <1 μm.

Measurement of cell volume

The volume of each cell was measured using serial images of optical sections according to the previous publication [63]. Three-dimensional imaging was performed for GFP fluorescence in the stably transfected cells at 1.05 μm z-axis intervals using a 63 × /1.2 oil-immersion lens on a confocal microscope LSM710 (Carl Zeiss). Cross-sectional area of the cell was calculated from each sectioned image using Fiji software, and cell volume was estimated as a sum of them.

Cell viability assay

As a hyperosmotic treatment, the S2 cells were exposed to the culture medium containing 0.4 M trehalose for 48 h. The cells were stained with Hoechst33342 (6.7 μg/mL, Lonza) and PI (0.67 μg/mL, Dojindo) for 30 min and observed with a fluorescence microscope BZ-X810 (Keyence). Hoechst33342-positive and PI-negative cells were counted as live cells and double-positive cells were counted as dead cells. The survival rates were calculated in 6 wells for each condition by counting 500 to 700 cells/well using analysis software Hybrid Cell Count (Keyence).

Estimation of the amount of endogenous CAHS3 protein by immunoblotting

After extensive washing with purer water, approximately 100 R. varieornatus were lysed using pestle in 30 μL PBS containing complete EDTA-free protease inhibitors (Roche) and centrifuged at 16,000 × g for 10 min. The soluble fractions of tardigrade lysate were mixed with 5 × SDS sample buffer (62.5 mM Tris-HCl (pH6.8), 25% glycerol, 10% sodium dodecyl sulfate, and 0.01% bromophenol blue) and 2-mercapto-ethanol. After heated at 100°C for 3 min, the samples were resolved by SDS-PAGE analysis and electroblotted onto PVDF membrane (Millipore). The membrane was blocked with 1% normal goat serum (Abcam) for 1 h at room temperature and reacted with the affinity-purified CAHS3 antibody diluted by 1% normal goat serum for 1 h at room temperature. After washed with TBS-T 3 times, the membrane was reacted with diluted peroxidase labeled anti-rabbit IgG antibody (KPL) for 1 h at room temperature. The membrane was washed with TBS-T 3 times, and then antibody-antigen complex was detected by ImageQuant LAS 500 (Cytiva) using enhanced chemiluminescence system (GE Healthcare). The diluted series of recombinant CAHS3 proteins (2.5, 5.0, 10.0, 20.0, 40.0 ng) were analyzed simultaneously on the same blot as quantification standards. Signal intensity of each corresponding band was measured by Fiji software and a linear regression was used to generate a standard curve between the signal intensity and the amount of protein as [Signal intensity] = [Amount of protein (ng)] × 446595.3952–696903.625; R² = 0.9962. Using the well-fitted standard curve, the amount of endogenous CAHS3 protein was calculated to be approximately 3.81 ng per tardigrade.