A sodium-dependent trehalose transporter contributes to anhydrobiosis in insect cell line, Pv11.

Pv11 is the only animal cell line that, when preconditioned with a high concentration of trehalose, can be preserved in the dry state at room temperature for more than one year while retaining the ability to resume proliferation. This extreme desiccation tolerance is referred to as anhydrobiosis. Here, we identified a transporter that contributes to the recovery of Pv11 cells from anhydrobiosis. In general, the solute carrier 5 (SLC5)-type secondary active transporters cotransport Na+ and carbohydrates including glucose. The heterologous expression systems showed that the transporter belonging to the SLC5 family, whose expression increases upon rehydration, exhibits Na+-dependent trehalose transport activity. Therefore, we named it STRT1 (sodium-ion trehalose transporter 1). We report an SLC5 family member that transports a naturally occurring disaccharide, such as trehalose. Knockout of the Strt1 gene significantly reduced the viability of Pv11 cells upon rehydration after desiccation. During rehydration, when intracellular trehalose is no longer needed, Strt1-knockout cells released the disaccharide more slowly than the parental cell line. During rehydration, Pv11 cells became roughly spherical due to osmotic pressure changes, but then returned to their original spindle shape after about 30 min. Strt1-knockout cells, however, required about 50 min to adopt their normal morphology. STRT1 probably regulates intracellular osmolality by releasing unwanted intracellular trehalose with Na+, thereby facilitating the recovery of normal cell morphology during rehydration. STRT1 likely improves the viability of dried Pv11 cells by rapidly alleviating the significant physical stresses that arise during rehydration.

Effective methods for immobilization of non-adherent Pv11 cells while maintaining their desiccation tolerance.

Pv11 was derived from embryos of the sleeping chironomid Polypedilum vanderplanki, which displays an extreme form of desiccation tolerance known as anhydrobiosis. Pre-treatment with a high concentration of trehalose allows Pv11 cells to enter anhydrobiosis. In the dry state, Pv11 cells preserve transgenic luciferase while retaining its activity. Thus, these cells could be utilized for dry-preserving antibodies, enzymes, signaling proteins or other valuable biological materials without denaturation. However, Pv11 cells grow in suspension, which limits their applicability; for instance, they cannot be integrated into microfluidic devices or used in devices such as sensor chips. Therefore, in this paper, we developed an effective immobilization system for Pv11 cells that, crucially, allows them to maintain their anhydrobiotic potential even when immobilized. Pv11 cells exhibited a very high adhesion rate with both biocompatible anchor for membrane (BAM) and Cell-Tak coatings, which have been reported to be effective on other cultured cells. We also found that Pv11 cells immobilized well to uncoated glass if handled in serum-free medium. Interestingly, Pv11 cells showed desiccation tolerance when trehalose treatment was done prior to immobilization of the cells. In contrast, trehalose treatment after immobilization of Pv11 cells resulted in a significant decrease in desiccation tolerance. Thus, it is important to induce anhydrobiosis before immobilization. In summary, we report the successful development of a protocol for the dry preservation of immobilized Pv11 cells. Supplementary Information: The online version contains supplementary material available at 10.1007/s10616-023-00592-0.

Identification of Genomic Safe Harbors in the Anhydrobiotic Cell Line, Pv11.

Genomic safe harbors (GSHs) provide ideal integration sites for generating transgenic organisms and cells and can be of great benefit in advancing the basic and applied biology of a particular species. Here we report the identification of GSHs in a dry-preservable insect cell line, Pv11, which derives from the sleeping chironomid, Polypedilum vanderplanki, and similar to the larvae of its progenitor species exhibits extreme desiccation tolerance. To identify GSHs, we carried out genome analysis of transgenic cell lines established by random integration of exogenous genes and found four candidate loci. Targeted knock-in was performed into these sites and the phenotypes of the resulting transgenic cell lines were examined. Precise integration was achieved for three candidate GSHs, and in all three cases integration did not alter the anhydrobiotic ability or the proliferation rate of the cell lines. We therefore suggest these genomic loci represent GSHs in Pv11 cells. Indeed, we successfully constructed a knock-in system and introduced an expression unit into one of these GSHs. We therefore identified several GSHs in Pv11 cells and developed a new technique for producing transgenic Pv11 cells without affecting the phenotype.

Cas9-mediated genome editing reveals a significant contribution of calcium signaling pathways to anhydrobiosis in Pv11 cells.

Pv11 is an insect cell line established from the midge Polypedilum vanderplanki, whose larval form exhibits an extreme desiccation tolerance known as anhydrobiosis. Pv11 itself is also capable of anhydrobiosis, which is induced by trehalose treatment. Here we report the successful construction of a genome editing system for Pv11 cells and its application to the identification of signaling pathways involved in anhydrobiosis. Using the Cas9-mediated gene knock-in system, we established Pv11 cells that stably expressed GCaMP3 to monitor intracellular Ca2+ mobilization. Intriguingly, trehalose treatment evoked a transient increase in cytosolic Ca2+ concentration, and further experiments revealed that the calmodulin-calcineurin-NFAT pathway contributes to tolerance of trehalose treatment as well as desiccation tolerance, while the calmodulin-calmodulin kinase-CREB pathway conferred only desiccation tolerance on Pv11 cells. Thus, our results show a critical contribution of the trehalose-induced Ca2+ surge to anhydrobiosis and demonstrate temporally different roles for each signaling pathway.