Optogenetic Investigations of the Role of the Actin-ATP Binding Site in Cofilin-Actin Rod Formation and the Stress-associated Responses of Actin-binding Proteins

Abstract

Cytoskeletal dysregulation is a hallmark of various neurodegenerative diseases, including Alzheimer's, Parkinson's, and Huntington's diseases, resulting in the formation of aberrant actin-rich structures such as cofilin-actin rods. These structures arise under conditions of oxidative and energetic stress, leading to synaptic loss and cellular dysfunction. Understanding the structural factors underlying the stress-associated interaction of cofilin and actin is crucial for elucidating the pathogenesis of neurodegenerative diseases. The actin-ATP interaction lies at the core of stress-induced cytoskeletal anomalies, with ATP depletion favoring the formation of cofilin-actin rods. While the contributions of actin's nucleotide binding residues to rod formation remain unclear, recent advancements, such as the CofActor optogenetic system, have shed light on this phenomenon. By mutating key ATP-binding residues in the actin-ATP interface, we elucidated their role in cytoskeletal anomalies under both homeostatic and stress conditions. Our findings highlight the importance of specific nucleotide-binding actin residues in the pathogenesis of neurodegenerative diseases, providing insights into potential therapeutic targets and its utility for experimentalists. In a related project, we investigated the ability of an optogenetic profilin to respond to applied cellular stress. Initial analysis into the ability of various actin-binding proteins to participate in the formation aberrant cytoskeletal structures such as actin-cofilin rods and stress granules as a result of energetic and oxidative stress, revealed that profilin has a differing response that isn't associated with actin, and instead associated with stress granules. Whether these cytoskeletal phenomena can be harnessed for the development of biosensors for cytoskeletal dysfunction and, by extension, neurodegenerative disease progression, remains an open question. In this project, we describe the design and development of an optogenetic iteration of profilin, an actin monomer binding protein with critical functions in cytoskeletal dynamics. We showed that the optically activated profilin ('OptoProfilin') can act as an optically triggered biosensor of applied cellular stress in select immortalized cell lines. Notably, OptoProfilin is a single component biosensor, likely increasing its utility for experimentalists.