Prof. Arnaud Gautier reports greenFAST and redFAST, orthogonal variants of FAST for multicolor imaging, in Nature Chemical Biology. A powerful tool for cell division monitoring, sequential or multiple protein-protein interaction studies, high-content analysis, and much more!
greenFAST and redFAST, a novel set of tags for multicolor imaging
The paper discloses various modalities of implementation of greenFAST & redFAST: two-color confocal microscopy, fluorescence lifetime imaging microscopy (FLIM), two-photon excitation, and even super-resolution microscopy (SOFI) with redFAST.
greenFAST & redFAST as cell cycle reporters in zebrafish embryos
greenFAST / redFAST labeling seems particularly promising for dynamic recording in vivo. The authors apply it to the FUCCI (fluorescence ubiquitination cell cycle indicator) in mammalian cells and in zebrafish embryos. In the latter, it allowed exquisite monitoring of individual nuclei from the 256-cell stage through to the tenth cycle. In addition, fluorescence recording of the whole embryo revealed proliferation patterns and asynchronic division as early as 256 cells. Detailed analysis of cell cycles as short as 15 min and as early as 3.5 hpf is only possible with quasi-instantaneous fluorescence as provided by FAST and its variants.
two-color protein-protein interaction reporters
splitFAST, earlier published in Nature Communications, is the only reversible fluorescence complementation reporter with rapid association/dissociation kinetics. Aiming breakthrough biosensors capable to report sequential or multiple protein interactions, the authors have hence developed split tags, namely split-greenFAST and split-redFAST. To illustrate their potential, they have tracked the formation of FKBP-FKBP dimers mediated by AP1510. And further, their exchange to FRB-FKBP dimers upon the addition of rapamycin.
They have thus proven the ability of split-greenFAST / split-redFAST to deliver facile readout of multiple or sequential protein interactions with good contrast and temporal resolution.