Mapping the Cellular Distribution of an Optogenetic Protein Using a Light-Stimulation Grid.

Our goal was to accurately track the cellular distribution of an optogenetic protein and evaluate its functionality within a specific cytoplasmic location. To achieve this, we co-transfected cells with nuclear-targeted cAMP sensors and our laboratory-developed optogenetic protein, bacterial photoactivatable adenylyl cyclase-nanoluciferase (bPAC-nLuc). bPAC-nLuc, when stimulated with 445 nm light or luciferase substrates, generates adenosine 3',5'-cyclic monophosphate (cAMP). We employed a solid-state laser illuminator connected to a point scanning system that allowed us to create a grid/matrix pattern of small illuminated spots (~1 µm(2)) throughout the cytoplasm of HC-1 cells. By doing so, we were able to effectively track the distribution of nuclear-targeted bPAC-nLuc and generate a comprehensive cAMP response map. This map accurately represented the cellular distribution of bPAC-nLuc, and its response to light stimulation varied according to the amount of protein in the illuminated spot. This innovative approach contributes to the expanding toolkit of techniques available for investigating cellular optogenetic proteins. The ability to map its distribution and response with high precision has far-reaching potential and could advance various fields of research.