The Power of Desiccation: A Study into Developing a Method to Screen Tardigrade Disordered Proteins for Phase Separation

Abstract

Tardigrades, also known as water bears, are microscopic organisms capable of surviving conditions such as extreme temperatures and deprivation from oxygen and water. Numerous studies have found that tardigrade intrinsically disordered proteins (TDPs), proteins without a fixed tertiary structure, are linked to tolerance to desiccation (removal of water). Some intrinsically disordered proteins undergo phase separation towards the formation of membraneless organelles within cells. In particular, tardigrade cytoplasmic abundant heat-soluble (CAHS) proteins were discovered to form non-crystalline amorphous solids via vitrification upon drying conditions. As the mechanisms by which TDPs operate during stress tolerance are not very well understood, the research done in this thesis sought to develop a method of screening TDPs for liquid phase separating interactions, which can then be utilized for the further understanding of the mechanisms the proteins are involved in. Greater understanding of desiccation tolerance mechanisms are of particular importance to xero-protection and astrobiology as harnessing desiccation tolerance can reduce global reliance on cold storage, provide mechanisms to confer resistance to toxic chemicals in microbial strains engineered to produce them, and provide technologies to improve biological resistance to extraterrestrial conditions. In order to develop a method to study the liquid phase separation interactions of TDPs, Yeast-2-Hybrid (Y2H) experiments were conducted on a library of five tardigrade cytoplasmic abundant heat soluble proteins (CAHS) to screen for protein interactions. These experiments were followed by integration of the CAHS protein library into a newly developed Yeast- Liquid-Hybrid (YLH) method that screened for liquid phase separation interactions. The Y2H experiments conducted in this paper found four positive protein-protein interactions among the CASH library and additionally contributed to the discovery of a CAHS protein capable of recruiting the RNA polymerase. The YLH experiments conducted on the same library of CAHS proteins found an additional eight potential phase separating interactions. While these results are promising, further experiments are necessary to confirm that the protein interactions observed in the YLH assays are the results of phase separation.