Stress-dependent cell stiffening by tardigrade tolerance proteins through reversible formation of cytoskeleton-like filamentous network and gel-transition

Abstract

Tardigrades are able to tolerate almost complete dehydration by entering a reversible ametabolic state called anhydrobiosis and resume their animation upon rehydration. Dehydrated tardigrades are exceptionally stable and withstand various physical extremes. Although trehalose and late embryogenesis abundant (LEA) proteins have been extensively studied as potent protectants against dehydration in other anhydrobiotic organisms, tardigrades produce high amounts of tardigrade-unique protective cytoplasmic-abundant heat-soluble (CAHS) proteins which are essential for the anhydrobiotic survival of tardigrades. However, the precise mechanisms of their action in this protective role are not fully understood. In the present study, we first postulated the presence of tolerance proteins that form protective condensates via phase separation in a stress-dependent manner and searched for tardigrade proteins that reversibly form condensates upon dehydration-like stress. Through comprehensive analysis, we identified 336 such proteins, collectively dubbed "dehydration-induced reversibly condensing proteins (DRPs)". Unexpectedly, we rediscovered CAHS proteins as highly enriched in DRPs, 3 of which were major components of DRPs. We revealed that these CAHS proteins reversibly polymerize into many cytoskeleton-like filaments depending on hyperosmotic stress in cultured cells and undergo reversible gel-transition in vitro, which increases the mechanical strength of cell-like microdroplets. The conserved putative helical C-terminal region is necessary and sufficient for filament formation by CAHS proteins, and mutations disrupting the secondary structure of this region impaired both the filament formation and the gel transition. On the basis of these results, we propose that CAHS proteins are novel cytoskeletal proteins that form filamentous networks and undergo gel-transition in a stress-dependent manner to provide on-demand physical stabilization of cell integrity against deformative forces during dehydration and contribute to the exceptional stability of dehydrated tardigrades.

The tardigrade protein CAHS D interacts with, but does not retain, water in hydrated and desiccated systems

“…Here the 1D4 tag serves as a control to ensure that mVenus is not introducing artifacts. Additionally, sorbitol was selected to allow for comparisons to previous work carried out with CAHS proteins in cells 43 , 49 . CAHS proteins are known to form hydrogels in a concentration-dependent fashion in vitro and are suspected to do the same within cells during osmotic shock due to the observation of fiber-like formation and stiffening in vivo 25 , 34 , 43 .…”

Sexual dimorphism in the tardigradeParamacrobiotus metropolitanustranscriptome

“…Interestingly, we observed sex-biased expression of several anhydrobiosis genes, hypothesized to play protective roles during anhydrobiosis (29-32, 90). For instance, CAHS genes are tardigrade-specific proteins that form gel filaments that possibly protect cells (91-93). Recent studies have observed tissue/organelle specificity for these proteins, which further implies the existence of orthologs with sex-specific expression (79).…”

Deciphering the Biological Enigma—Genomic Evolution Underlying Anhydrobiosis in the Phylum Tardigrada and the Chironomid Polypedilum vanderplanki

“…Although these two protein families do not show sequence similarity with canonical LEA proteins, they both form alpha-helical structures under water-deficit conditions or upon TFE treatment, as reported for LEA proteins. Moreover, since 2021, multiple studies have reported the formation of higher-order structures by CAHS proteins [ 92 , 93 , 94 , 95 , 96 ]. These studies argue that purified recombinant CAHS proteins form fiber-like aggregates or gel-like lumps at high concentrations, as is assumed to occur with the onset of desiccation.…”