Interaction of iLID Optogenetic Proteins Characterized using 3D Single-Molecule Tracking in Live E.coli

Single-molecule fluorescence microscopy is a powerful tool that can be used to resolve cellular structures with nanometers of resolution. By localizing molecules with high precision over time, protein motion can be measured and used to resolve different diffusive states. Diffusive states are assigned to protein complexes using computational models with significant statistical power, and require biological validation which is often deliberatively perturbative to cellular function. In the work presented in this dissertation, I explore incorporating a non-invasive, transient method for disrupting protein localization and prospective diffusive states. I utilize light-induced dimerization domains to transiently sequester proteins to non-native cellular compartments upon light stimulation. To test the feasibility of this method, I have characterized the optical response of the improved Light Induced Dimerization (iLID) optogenetic system using conventional imaging methods in addition to single-molecule tracking experiments. To carefully examine the iLID system dynamics, I have extended our single-molecule analysis workflow to incorporate trajectory simulations of membrane-associated molecules. Further, I have added a full-trajectory analysis module that identifies changes in diffusion rate to quantify residence times of single-molecules at binding sites, and the kinetics of the interaction. Through these analyses, I have shown that the iLID system can be activated by longer wavelengths that are minimally absorbed in in vitro conditions. Further, I have identified transient interactions of the iLID optogenetic proteins that are not detectable in diffraction-limited imaging. This analysis highlights areas for characterization and improvement of the iLID optical response.