Date on Master's Thesis/Doctoral Dissertation
Committee Co-Chair (if applicable)
ARID proteins; LEA proteins; desiccation tolerance; artemia franciscana; polypedilum vanderplanki; trehalose
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.
Belott, Clinton J., "Visualizing Anhydrobiosis: Liquid-liquid phase separation, membraneless organelles, and cellular reorganization." (2021). Electronic Theses and Dissertations. Paper 3778.