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Visualize and Manipulate Protein Activity in Living Cells

Signaling circuits have complex nested feedback loops and are tightly regulated in time and space. The ability to visualize the interactome and genetically reprogram the signaling components is critical to dissect entangled signaling pathways. My presentation will mainly cover two topics. First, we developed  binder/tag , a generalizable approach that is both convenient for general (FRET) biosensor engineering and sensitive enough to follow single molecule activity in living cells. We described different  binder/tag analogs (tagSrc, tagFyn, tagSyk, tagFAK), and an orthogonal binder/tag pair. SPT showed slowly diffusing islands of activated Src within Src clusters and dynamics of activation in adhesions. Quantitative analysis and stochastic modeling revealed in vivo Src kinetics. Second, we developed Z-lock, an optogenetic approach for reversible, light-controlled steric inhibition of protein active sites. Computer-assisted protein design was used to optimize linkers and Zdk-LOV affinity, for both effective binding in the dark, and effective light-induced release of the intramolecular interaction. Z-lock cofilin was shown to have filament actin severing ability in vitro, and in living cancer cells, it produced protrusions and invadopodia in response to light. An active fragment of the tubulin acetylase αTAT was similarly modified and shown to acetylate tubulin upon irradiation. Ultimately, combining these two techniques will allow me to directly control one protein and visualize the activity change of the others.