Membraneless and membrane-bound organelles in an anhydrobiotic cell line are protected from desiccation-induced damage.

Anhydrobiotic species can survive virtually complete water loss by entering a reversible ametabolic glassy state that may persist for years in ambient conditions. The Pv11 cell line was derived from the egg mass of the anhydrobiotic midge, Polypedilum vanderplanki, and is currently the only available anhydrobiotic cell line. Our results demonstrate that the necessary preconditioning for Pv11 cells to enter anhydrobiosis causes autophagy and reduces mitochondrial respiration by over 70%. We speculate that reorganizing cellular bioenergetics to create and conserve energy stores may be valuable to successfully recover after rehydration. Furthermore, mitochondria in preconditioned cells lose their membrane potential during desiccation but rapidly restore it within 30 min upon rehydration, demonstrating that the inner mitochondrial membrane integrity is well-preserved. Strikingly, the nucleolus remains visible immediately upon rehydration in preconditioned cells while absent in control cells. In contrast, a preconditioning-induced membraneless organelle reformed after rehydration, demonstrating that membraneless organelles in Pv11 cells can be either stabilized or recovered. Staining the endoplasmic reticulum and the Golgi apparatus revealed that these organelles fragment during preconditioning. We hypothesize that this process reduces sheering stress caused by rapid changes in cellular volume during desiccation and rehydration. Additionally, preconditioning was found to cause the filamentous-actin (F-actin) network to disassemble significantly and reduce the fusion of adjacent plasma membranes. This study offers several exciting avenues for future studies in the animal model and Pv11 cell line that will further our understanding of anhydrobiosis and may lead to advancements in storing sensitive biologics at ambient temperatures for months or years.

New insights into anhydrobiosis using cellular dielectrophoresis-based characterization.

Late embryogenesis abundant (LEA) proteins are found in desiccation-tolerant species from all domains of life. Despite several decades of investigation, the molecular mechanisms by which LEA proteins confer desiccation tolerance are still unclear. In this study, dielectrophoresis (DEP) was used to determine the electrical properties of Drosophila melanogaster (Kc167) cells ectopically expressing LEA proteins from the anhydrobiotic brine shrimp, Artemia franciscana. Dielectrophoresis-based characterization data demonstrate that the expression of two different LEA proteins, AfrLEA3m and AfrLEA6, increases cytoplasmic conductivity of Kc167 cells to a similar extent above control values. The impact on cytoplasmic conductivity was surprising, given that the concentration of cytoplasmic ions is much higher than the concentrations of ectopically expressed proteins. The DEP data also supported previously reported data suggesting that AfrLEA3m can interact directly with membranes during water stress. This hypothesis was strengthened using scanning electron microscopy, where cells expressing AfrLEA3m were found to retain more circular morphology during desiccation, while control cells exhibited a larger variety of shapes in the desiccated state. These data demonstrate that DEP can be a powerful tool to investigate the role of LEA proteins in desiccation tolerance and may allow to characterize protein-membrane interactions in vivo, when direct observations are challenging.