DESIGN OF PDRONPA1.2-BASED OPTOGENETIC SWITHC FOR CONTROL OF DISEASE- ASSOCIATED CYTOSKELETAL STRUCTURES

Abstract

Actin is a protein present in all eukaryotic cells and is essential in muscle contraction and cell movement and structure. In order for actin filaments to function properly, it is important for them to be able to bind to ATP. However, stressful conditions can convert actin to a primarily ADP bound state which can alter its normal function and even cause the formation of actin-cofilin rods. These rods are a common feature in neurons of those who suffer from neurodegenerative disease such as Alzheimer's, Parkinson’s, and Huntington’s disease. Additionally, actin post-translational modifications are hypothesized to play a potential role in disease brains. Although there is a correlation between malformation of actin and neurodegenerative disease, not much is known about the formation of these structures and the role they play under stressful conditions. Previously, research on this topic has been conducted using the CofActor (Cofilin Actin optically responsive) system, which allows insight into the clustering of actin and cofilin through light activation. To analyze the behavior of these clusters independent of stress conditions, mutations were made at the ATP interaction sites. Using PCR amplification, sequencing, and widefield fluorescence microscopy, it was noted that three mutations had notable formations of actin-cofilin rods and inclusions, even in absence of energetic stress. To gain more knowledge on the behavior of actin before clumping, we incorporated the pDronpa1 optogenetic system instead, which allows for a wider range of experimentation due to its photocaging abilities. In other words, the pDronpa1 homodimer surrounds the actin in the dark, restricting its interaction with other actin monomers. Once it is light activated, the pDronpa1 dimer will dissociate allowing for formation of the actin clusters and for a clearer analysis of cytoskeletal dynamics in neurons. Experiments conducted so far have proven the photoswitchable capabilities of the pDronpa1 vector.