The impact of the Drosophila experimental system on studies of modern biology cannot be understated. The ability to tag endogenously expressed proteins is essential to maximize the use of this model organism. Here, we describe a method for labeling endogenous proteins with self-complementing split fluorescent proteins (split FPs) in a cell-type-specific manner in Drosophila A short fragment of an FP coding sequence is inserted into a specific genomic locus while the remainder of the FP is expressed using an available GAL4 driver line. In consequence, complementation fluorescence allows examination of protein localization in particular cells. Besides, when inserting tandem repeats of the short FP fragment at the same genomic locus, we can substantially enhance the fluorescence signal. The enhanced signal is of great value in live-cell imaging at the subcellular level. We can also accomplish a multicolor labeling system with orthogonal split FPs. However, other orthogonal split FPs do not function for in vivo imaging besides split GFP. Through protein engineering and in vivo functional studies, we report a red split FP that we can use for duplexed visualization of endogenous proteins in intricate Drosophila tissues. Using the two orthogonal split FP systems, we have simultaneously imaged proteins that reside in distinct subsynaptic compartments. Our approach allows us to study the proximity between and localization of multiple proteins endogenously expressed in essentially any cell type in Drosophila.
Keywords: Drosophila; fluorescence microscopy; fluorescent protein; neuron; tissue.