Date on Master's Thesis/Doctoral Dissertation

12-2021

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Biology

Degree Program

Biology, PhD

Committee Chair

Menze, Michael A.

Committee Member

Schultz, David J.

Committee Member

Running, Mark P.

Committee Member

Uversky, Vladimir N.

Committee Member

Crowe, John H.

Author's Keywords

Late embryogenesis abundant proteins; phase separation; artemia franciscana; dehydration; stress tolerance; preservation

Notes

This work outlines the characterization of proteins associated with dehydration tolerance and long-term desiccation tolerance across several taxonomic groups of animals. A focus on proteins from the primitive brine shrimp, Artemia franciscana, is used to investigate the properties of different families of LEA proteins. This work includes research into liquid-liquid phase separation of LEA proteins.

Abstract

Anhydrobiosis is the remarkable phenomenon of “life without water”. It is a common technique found in plant seeds, and a rare technique utilized by some animals to temporarily stop the clock of life and enter a stasis for up to several millennia by removing all of their cellular water. If this phenomenon can be replicated, then biological and medical materials could be stored at ambient temperatures for centuries, which would address research challenges as well as enhance the availability of medicine in areas of the world where refrigeration, freezing, and cold-chain infrastructure are not developed or infeasible. Furthermore, modifying crop tissues could make them resistant to droughts, addressing one of the greatest threats to food stability around the world. This work utilizes a combination of computational techniques and novel approaches to performing biochemistry without water to elucidate the mechanisms of function of specialized proteins that are responsible for anhydrobiosis in animals, particularly the anhydrobiotic cysts of the brine shrimp Artemia franciscana. A detailed evaluation of the chemical properties of anhydrobiosis-related, intrinsically disordered proteins indicates that there are multiple protein-based strategies to achieve anhydrobiosis, but that late embryogenesis abundant (LEA) proteins are the most well understood. However, the mechanisms of LEA protein function have never been demonstrated, resulting in a wide variety of hypotheses regarding their ability to confer desiccation tolerance. This work demonstrates that a group 1 LEA protein, AfLEA1.1, and a group 6 LEA protein, AfrLEA6, undergo liquid-liquid phase separations during desiccation and thereby transiently form novel protective membraneless organelles which partition specific proteins and nucleic acids. These desiccation-induced cellular compartments are a novel mechanism to explain how LEA proteins confer desiccation tolerance, and the drivers of this behavior have been linked to the consensus sequences that define these LEA proteins. Therefore, the separation of aqueous proteins into a specialize compartment during drying is unlikely to only be a function of AfLEA1.1 and AfrLEA6, but actually the mechanism by which group 1 and group 3 LEA proteins function in plant seeds and anhydrobiotic animals. These results indicate that when water is unavailable, anhydrobiotic organisms substitute it with their own solvents.

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