Significance: Recent research has shown that intracellular proteinaceous condensates (membraneless organelles [MLOs]) are involved in various processes, ranging from Alzheimer’s disease to RNA processing, and here we demonstrate that this phenomenon governs a mechanism of anhydrobiosis. The proteinAfrLEA6 is found in the desiccation-tolerant life stage of the animal extremophileArtemia franciscana,and the protein engages in two distinct molecular mechanisms to confer protection during water loss.AfrLEA6 forms MLOs that may act as protective nodes for desiccation-sensitive proteins, while a cytosolic fraction of the protein promotes structural integrity of cells during anhydrobiosis. These findings significantly advance our understanding of “life without water” and promote transformative advancements in various fields, ranging from cell preservation technology to improvement of crop desiccation tolerance.

Group 1 (Dur-19, PF00477, LEA_5) Late Embryogenesis Abundant (LEA) proteins are present in organisms from all three domains of life, Archaea, Bacteria, and Eukarya. Surprisingly, Artemia is the only genus known to include animals that express group 1 LEA proteins in their desiccation-tolerant life-history stages. Bioinformatics analysis of circular dichroism data indicates that the group 1 LEA protein AfLEA1 is surprisingly ordered in the hydrated state and undergoes during desiccation one of the most pronounced disorder-to-order transitions described for LEA proteins from A. franciscana. The secondary structure in the hydrated state is dominated by random coils (42%) and β-sheets (35%) but converts to predominately α-helices (85%) when desiccated. Interestingly, AfLEA1 interacts with other proteins and nucleic acids, and RNA promotes liquid–liquid phase separation (LLPS) of the protein from the solvent during dehydration in vitro. Furthermore, AfLEA1 protects the enzyme lactate dehydrogenase (LDH) during desiccation but does not aid in restoring LDH activity after desiccation-induced inactivation. Ectopically expressed in D. melanogaster Kc167 cells, AfLEA1 localizes predominantly to the cytosol and increases the cytosolic viscosity during desiccation compared to untransfected control cells. Furthermore, the protein formed small biomolecular condensates in the cytoplasm of about 38% of Kc167 cells. These findings provide additional evidence for the hypothesis that the formation of biomolecular condensates to promote water stress tolerance during anhydrobiosis may be a shared feature across several groups of LEA proteins that display LLPS behaviors.

Water is an integral and necessary component of life. It is then, exceedingly remarkable that some species are capable of surviving virtually complete water loss for extended periods of time. Several decades of intense research into anhydrobiosis, or life without water, have given significant insights into the molecular mechanisms governing this phenomenon. Anhydrobiosis-related intrinsically disordered (ARID) proteins have been demonstrated to be critically important for desiccation tolerance in many anhydrobiotic species and exhibit a considerably wide range of protective properties that include membrane stabilization, reinforcing bioglass formation, and protein stabilization. This dissertation begins with cellular dielectrophoresis suggesting that two ARID proteins, AfrLEA3m and AfrLEA6, were undergoing significant folding in vivo due to moderate intracellular water loss. This was hypothesized to result in these ARID proteins changing from an untangled state to a tangled one, thereby increasing intracellular viscosity. This dissertation then proceeds to further explore AfrLEA6 in vivo, finding it able to undergo a domain-dependent liquid-liquid phase separation to form a selective, stress granule-like membraneless organelle (MLO). Furthermore, the dilute fraction of AfrLEA6 was found to rapidly increase intracellular viscosity at moderate levels of intracellular water loss, supporting the hypothesis that AfrLEA6 was entering a tangled state. Lastly, this dissertation explores cellular reorganization in the anhydrobiotic Polypedilum vanderplanki Pv11 cell line as it undergoes preconditioning, desiccation, and rehydration. The nucleus, mitochondria, ER, Golgi apparatus, nucleolus, F-actin network, and plasma membrane all demonstrated significant morphological and/or physiological changes in response to preconditioning, desiccation and/or rehydration. Surprisingly, the nucleolus still appeared assembled immediately after rehydration, while an identified stress-induced MLO required 1 h to reassemble, suggesting that these two MLOs were protected by different mechanisms. Altogether, this dissertation describes how an ARID protein could protect a wide variety of targets without necessarily requiring a high ratio of protective protein to targets. Furthermore, this dissertation describes the complex cellular reorganization that occurs in Pv11 during preconditioning, desiccation, and rehydration, which may help guide future experiments in the animal model to further our understanding of anhydrobiosis.