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 proteins. Cytoplasmic-abundant heat-soluble (CAHS) proteins are uniquely invented in the lineage of eutardigrades, a major class of the phylum Tardigrada and are essential for their anhydrobiotic survival. 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 a comprehensive search using a desolvating agent, trifluoroethanol (TFE), we identified 336 proteins, collectively dubbed “TFE-Dependent ReversiblY condensing Proteins (T-DRYPs).” Unexpectedly, we rediscovered CAHS proteins as highly enriched in T-DRYPs, 3 of which were major components of T-DRYPs. 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. Furthermore, CAHS proteins increased cell stiffness in a hyperosmotic stress-dependent manner and counteract the cell shrinkage caused by osmotic pressure, and even improved the survival against hyperosmotic stress. 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 cytoskeleton-like 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 could contribute to the exceptional physical stability in a dehydrated state.

Navigation of fast migrating cells such as amoeba Dictyostelium and immune cells are tightly associated with their morphologies that range from steady polarized forms that support high directionality to those more complex and variable when making frequent turns. Model simulations are essential for quantitative understanding of these features and their origins, however systematic comparisons with real data are underdeveloped. Here, by employing deep-learning-based feature extraction combined with phase-field modeling framework, we show that a low dimensional feature space for 2D migrating cell morphologies obtained from the shape stereotype of keratocytes, Dictyostelium and neutrophils can be fully mapped by an interlinked signaling network of cell-polarization and protrusion dynamics. Our analysis links the data-driven shape analysis to the underlying causalities by identifying key parameters critical for migratory morphologies both normal and aberrant under genetic and pharmacological perturbations. The results underscore the importance of deciphering self-organizing states and their interplay when characterizing morphological phenotypes.

In fast-moving cells such as amoeba and immune cells, dendritic actin filaments are spatiotemporally regulated to shape large-scale plasma membrane protrusions. Despite their importance in migration, as well as in particle and liquid ingestion, how their dynamics are affected by micrometer-scale features of the contact surface is still poorly understood. Here, through quantitative image analysis of Dictyostelium on microfabricated surfaces, we show that there is a distinct mode of topographical guidance directed by the macropinocytic membrane cup. Unlike other topographical guidance known to date that depends on nanometer-scale curvature sensing protein or stress fibers, the macropinocytic membrane cup is driven by the Ras/PI3K/F-actin signaling patch and its dependency on the micrometer-scale topographical features, namely PI3K/F-actin–independent accumulation of Ras-GTP at the convex curved surface, PI3K-dependent patch propagation along the convex edge, and its actomyosin-dependent constriction at the concave edge. Mathematical model simulations demonstrate that the topographically dependent initiation, in combination with the mutually defining patch patterning and the membrane deformation, gives rise to the topographical guidance. Our results suggest that the macropinocytic cup is a self-enclosing structure that can support liquid ingestion by default; however, in the presence of structured surfaces, it is directed to faithfully trace bent and bifurcating ridges for particle ingestion and cell guidance.