Open AccessOrthogonal fluorescent chemogenetic reporters for multicolor imaging
Author(s) -
Alison G. Tebo,
Benjamien Moeyaert,
Marion Thauvin,
Irene Carlón-Andrés,
Dorothea Böken,
Michel Volovitch,
Sergi PadillaParra,
Peter Dedecker,
Sophie Vriz,
Arnaud Gautier
Publication year - 2020
Publication title -
nature chemical biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 6.412
H-Index - 216
eISSN - 1552-4469
pISSN - 1552-4450
DOI - 10.1038/s41589-020-0611-0
Subject(s) - fluorescence , bimolecular fluorescence complementation , fluorophore , chromophore , orthogonality , live cell imaging , fluorescence microscope , fluorescent protein , cell function , absorption (acoustics) , fluorescence lifetime imaging microscopy , biophysics , nanotechnology , cell , chemistry , green fluorescent protein , microbiology and biotechnology , biology , materials science , optics , biochemistry , physics , photochemistry , mathematics , gene , geometry
Spectrally separated fluorophores allow the observation of multiple targets simultaneously inside living cells, leading to a deeper understanding of the molecular interplay that regulates cell function and fate. Chemogenetic systems combining a tag and a synthetic fluorophore provide certain advantages over fluorescent proteins since there is no requirement for chromophore maturation. Here, we present the engineering of a set of spectrally orthogonal fluorogen-activating tags based on the fluorescence-activating and absorption shifting tag (FAST) that are compatible with two-color, live-cell imaging. The resulting tags, greenFAST and redFAST, demonstrate orthogonality not only in their fluorogen recognition capabilities, but also in their one- and two-photon absorption profiles. This pair of orthogonal tags allowed the creation of a two-color cell cycle sensor capable of detecting very short, early cell cycles in zebrafish development and the development of split complementation systems capable of detecting multiple protein-protein interactions by live-cell fluorescence microscopy.