Multiplexed In Vivo Imaging with Fluorescence Lifetime-Modulating Tags.

Fluorescence lifetime imaging microscopy (FLIM) opens new dimensions for highly multiplexed imaging in live cells and organisms using differences in fluorescence lifetime to distinguish spectrally identical fluorescent probes. Here, a set of fluorescence-activating and absorption-shifting tags (FASTs) capable of modulating the fluorescence lifetime of embedded fluorogenic 4-hydroxybenzylidene rhodanine (HBR) derivatives is described. It is shown that changes in the FAST protein sequence can vary the local environment of the chromophore and lead to significant changes in fluorescence lifetime. These fluorescence lifetime-modulating tags enable multiplexed imaging of up to three targets in one spectral channel using a single HBR derivative in live cells and live zebrafish larvae. The combination of fluorescence lifetime multiplexing with spectral multiplexing allows to successfully image six targets in live cells, opening great prospects for multicolor fluorescence lifetime multiplexing.

Improving Split Reporters of Protein-Protein Interactions through Orthology-Based Protein Engineering.

Protein-protein interactions (PPIs) can be detected through selective complementation of split fluorescent reporters made of two complementary fragments that reassemble into a functional fluorescent reporter when in close proximity. We previously introduced splitFAST, a chemogenetic PPI reporter with rapid and reversible complementation. Here, we present the engineering of splitFAST2, an improved reporter displaying higher brightness, lower self-complementation, and higher dynamic range for optimal monitoring of PPI using an original protein engineering strategy that exploits proteins with orthology relationships. Our study allowed the identification of a system with improved properties and enabled a better understanding of the molecular features controlling the complementation properties. Because of the rapidity and reversibility of its complementation, its low self-complementation, high dynamic range, and improved brightness, splitFAST2 is well suited to study PPI with high spatial and temporal resolution, opening great prospects to decipher the role of PPI in various biological contexts.

Engineering of Tunable Allosteric-like Fluorogenic Protein Sensors.

Optical protein sensors that enable detection of relevant biomolecules of interest play central roles in biological research. Coupling fluorescent reporters with protein sensing units has enabled the development of a wide range of biosensors that recognize analytes with high selectivity. In these sensors, analyte recognition induces a conformational change in the protein sensing unit that can modulate the optical signal of the fluorescent reporter. Here, we explore various designs for the creation of tunable allosteric-like fluorogenic protein sensors through incorporation of sensing protein units within the chemogenetic fluorescence-activating and absorption-shifting tag (FAST) that selectively binds and stabilizes the fluorescent state of 4-hydroxybenzylidene rhodanine (HBR) analogs. Conformational coupling allowed us to design analyte-responsive optical protein sensors through allosteric-like modulation of fluorogen binding.

A fluorogenic chemically induced dimerization technology for controlling, imaging and sensing protein proximity.

Molecular tools enabling the control and observation of the proximity of proteins are essential for studying the functional role of physical distance between two proteins. Here we present CATCHFIRE (chemically assisted tethering of chimera by fluorogenic-induced recognition), a chemically induced proximity technology with intrinsic fluorescence imaging and sensing capabilities. CATCHFIRE relies on genetic fusion to small dimerizing domains that interact upon addition of fluorogenic inducers of proximity that fluoresce upon formation of the ternary assembly, allowing real-time monitoring of the chemically induced proximity. CATCHFIRE is rapid and fully reversible and allows the control and tracking of protein localization, protein trafficking, organelle transport and cellular processes, opening new avenues for studying or controlling biological processes with high spatiotemporal resolution. Its fluorogenic nature allows the design of a new class of biosensors for the study of processes such as signal transduction and apoptosis.

Isolating and Engineering Fluorescence-Activating Proteins Using Yeast Surface Display.

This protocol describes the workflow to isolate and engineer fluorescence-activating proteins by yeast surface display. Fluorescence-activating proteins are an emerging class of fluorescent chemogenetic reporters for monitoring gene expression and protein localization in living cells and organisms. They become fluorescent upon binding exogenously applied fluorogenic organic dyes. Efficient fluorescence-activating proteins can be selected from yeast-displayed libraries by iterative rounds of fluorescence-activated cell sorting. The overall strategy is described, as well as a strategy for characterizing the affinity and spectroscopic properties of the selected clones.