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Cold stress is an important threat in the life history of fish. However, current research on the tolerance mechanisms of fish to cold stress is incomplete. To explore the relevant molecular mechanisms enabling cold stress tolerance in fish, here we studied ZF4 cells subjected to short-term (4 days) low temperature stress and long-term (3 months) low temperature acclimation. The results showed that cell viability decreased and the cytoskeleton shrank under short-term (4 days) low temperature stress, while the cell viability and the cytoskeleton became normal after cold acclimation at 18°C for 3 months. Further, when the cells were transferred to the lower temperature (13°C), the survival rate was higher in the acclimated than non-acclimated group. By investigating the oxidative stress pathway, we found that the ROS (reactive oxygen species) content increased under short-term (4 days) cold stress, coupled with changes in glutathione (GSH), catalase (CAT), superoxide dismutase (SOD) enzyme activity levels. In addition, overproduction of ROS disrupted physiological cellular homeostasis that generated apoptosis via the activation of the mitochondrial pathway. However, when compared with the non-domesticated group, both ROS levels and apoptosis were lowered in the long-term (3 months) domesticated cells. Taken together, these findings suggest that cold acclimation can improve the low temperature tolerance of the cells. This exploration of the mechanism by which zebrafish cells tolerate cold stress, thus contributes to laying the foundation for future study of the molecular mechanism of cold adaptation in fish.

Adhesion G protein-coupled receptors (aGPCRs) are a large family within the superfamily of G protein-coupled receptors involved in various physiological processes. One unique feature of aGPCRs is their long N-terminal extracellular regions (ECRs), which contain adhesive domains and a GPCR autoproteolysis-inducing (GAIN) domain. This GAIN domain promotes autoproteolytic cleavage of aGPCRs into N- and C-terminal fragments (NTF, CTF, respectively) after receptor biosynthesis. aGPCR signaling involves an interplay between the NTF and CTF that can be mechanically activated or modulated. However, how force affects the conformation/structure of the GAIN domain as a central structural element in aGPCR activation remains largely unknown. In this study, we investigated the mechanical stability of the GAIN domains of three aGPCRs from subfamilies B, G and L at a loading rate of 1 pN/s. Our findings demonstrate that the GAIN domains can be destabilized by forces from a few to 20 piconewtons (pN). Specifically, for the autocleaved aGPCRs, ADGRG1/GPR56 and ADGRL1/Latrophilin-1, forces over this range can cause detachment of the GAIN domain from the membrane-proximal Stachel element, which serves as an endogenous tethered agonist to aGPCRs, typically preceded with GAIN domain unfolding. For the non-cleavable aGPCR ADGRB3/BAI3, the GAIN domain undergoes complex mechanical unfolding over a similar force range. We also demonstrate that detachment of the GAIN domain can take place during cell migration, provided that the linkage between aGPCR and extracellular matrix is sufficiently stable. These results suggest that both structural stability of the GAIN domain and NTF/CTF dissociation are sensitive to physiological ranges of tensile forces, providing insights into the mechanical activation hypothesis of aGPCRs.