Using Photoswitching FRET to Define the Interaction Boundaries between the Rab1b GTPase and Secretory Cargo

FRET is a powerful tool to simultaneously establish and localize interactions between fluorescently tagged proteins with high spatial resolution. Rainey K.H. and Patterson G.H. introduced Photoswitching FRET (psFRET) using the Dronpa Photoswitching fluorescent protein. We present a straightforward detailed method, and a powerful software tool that allows adaptation of psFRET to diverse experimental setups. Image stacks, recording the decay of the Dronpa donor, serve as input to the software utility that includes effective preprocessing options preceding the calculation FRET efficiency at the single pixel level. We applied psFRET to generate interaction maps analyzing diverse interactions between cargo proteins, the GTPase Rab1b, and GRASP65 during ER to Golgi trafficking. Cargo-Rab1b interactions were restricted to the transit period from ER to Golgi. These data lend support to a mechanism whereby cargo sensing may regulate the level of downstream effectors recruitment to secretory membranes by Rab1.