LILAC: Designing and Implementing a Light-Sensitive Actin-Binding Peptide

Cytoskeletal structure and dynamics are key for cells to function. Not only does the cytoskeleton give static support to the cell, its self-organization and restructuring are critical for cytokinesis, muscle movement, lamellipodia driven cell movement and more. Visualization of the actin cytoskeleton is necessary for various fields of biological research, and there are several actin labels available. For live-cell imaging, the most popular label is Lifeact, a 17 amino-acid peptide derived from actin binding protein (ABP) 140 in yeast. Despite its use in over 7,000 studies, Lifeact has several known concentration-dependent side effects, likely due to its competitive binding with important ABPs, cofilin and myosin. I addressed this problem by creating a light-sensitive version of Lifeact that has low affinity in the dark, but high affinity for actin when excited by blue light. Using previous designs of optogenetic tools as a guide, I designed LILAC (Light-Induced Label for ACtin) by strategically integrating Lifeact into the reversibly unfolded J$\alpha$ helix of AsLOV2, a photosenstive protein from oat. Reversible, light-induced recruitment of LILAC to filamentous actin was validated both in live cells and with purified proteins. Pre-illumination subtraction and correlative imaging allow for enhanced visualization of actin, and the recovery time constant can be modulated with buffer conditions. I also utilized LILAC to pattern actin filaments \textit{in vitro}, a tool that could be applied to elucidate how components of the actin cytoskeleton are integrated spatiotemporally to form complex structures. In my thesis, I describe the development and application of LILAC as an imaging and patterning tool, though a wide array of other applications remain.