Elucidation of subcellular signaling networks by multiparameter imaging is hindered by a lack of sensitive FRET pairs spectrally compatible with the classic CFP/YFP pair. Here, we present a generic strategy to enhance the traditionally poor sensitivity of red FRET sensors by developing self-associating variants of mOrange and mCherry that allow sensors to switch between well-defined on- and off states. Requiring just a single mutation of the mFruit domain, this new FRET pair improved the dynamic range of protease sensors up to 10-fold and was essential to generate functional red variants of CFP-YFP-based Zn(2+) sensors. The large dynamic range afforded by the new red FRET pair allowed simultaneous use of differently colored Zn(2+) FRET sensors to image Zn(2+) over a broad concentration range in the same cellular compartment.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.