Fluorescent reporters and sensors play a central role in biological and medical research. Targeted to specific biomolecules or cells, they allow non-invasive imaging of the mechanisms that govern cells and organisms in real time. Recently, chemogenetic reporters composed of organic chromophores interacting with a protein moiety have challenged the hegemony of fluorescent proteins classically used in Cell Biology. Combining the advantage of synthetic fluorophores with the targeting selectivity of genetically encoded systems, these chemogenetic reporters open new perspectives for the study of cellular processes. With the support of the ERC consolidator grant, we have developed a new class of chemogenetic reporters, called FASTs (fluorescence-activating and absorption-shifting tags), which allow the visualization of gene expression and protein localization in living cells and organisms. Engineered using a concerted strategy of molecular engineering and directed evolution, FASTs are small protein tags that bind and stabilize the fluorescent state of fluorogenic chromophores. Dark when free in solution or cells, these so-called fluorogens allow the imaging of FAST-tagged proteins with very high contrast without the need for washing. FAST and its variants have proved to be a useful tool that is compatible with multiple microscopy modalities, including super-resolution microscopy, and model organisms. In particular, they excel in applications in oxygen-poor environments or in situations where the lack of delay in formation of a fluorescent complex allows the detection of rapid biological events. The modular nature of these reporters allowed us furthermore to design biosensors for the measurement of key metabolites. Finally, bisection of FASTs into two complementary fragments enabled the design of split fluorescent reporters with unprecedented rapid and reversible complementation, allowing imaging of dynamic protein-protein interactions with high spatial and temporal resolution. This strategy has been then extended to the generation of a tripartite split-FAST, enabling to study the proximity of three interactions, opening great prospects for studying the functions of multi-partner protein complexes. In addition to molecular imaging tools, we developed chemogenetic tools to control cellular functions. The specificity of cellular functions results from the spatial and temporal organization of functionally interacting proteins. Various strategies are used by cells to achieve specificity including protein compartmentalization in organelles, protein colocalization on membranes, or assembly of protein complexes mediated by specific scaffolds. Such spatial organization enables to increase effective molarity in biochemical processes and is essential for key cellular processes such as gene regulation, protein transport, organelle transport and positioning, signal transduction, metabolism, immune response or cell-cell communications. To study and understand the role of the spatiotemporal organization of proteins in these processes, we developed CATCHFIRE (Chemically Assisted Tethering of CHimera by Fluorogenic Induced Recognition) to control the physical proximity of proteins, and quantify this proximity. This tool relies on the genetic fusion of two small dimerization domains that can interact together in presence of a fluorogenic inducer of dimerization that fluoresces upon formation of the ternary assembly, allowing real-time monitoring of chemically induced proximity.
