Periodic Reporting for period 5 - FLUOSWITCH (Pushing the frontiers of biological imaging with genetically encoded fluorescence switches) Reporting period: 2022-04-01 to 2023-04-30 Summary of the context and overall objectives of the project Cells and organisms are complex machines driven by a set of dynamic biological events tightly orchestrated in space and time. The comprehensive molecular understanding of their inner workings requires acute molecular tools to observe and modulate the key triggers and cell signaling events, and ultimately identify novel therapeutic strategies. To study the molecular mechanisms that govern cells and organisms, we combined chemistry with protein engineering and genetic tools to develop novel approaches for the quantitative imaging and acute modulation of individual small molecules, proteins, organelles or cells over different scales in space and time. The project focused on the development of molecular chemogenetic reporters, sensors and actuators for monitoring and controlling the function of proteins and the behavior of cells. These tools can enable to address questions ranging from fundamental mechanisms to the causes of disease and the development of novel therapeutics. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far 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. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) Fluorescence microscopy has become the method of choice for characterizing cellular structures and biomolecules with high spatial and temporal resolution in living cells and organisms. In this project, we expanded the fluorescence toolbox with Fluorescence-Activating and absorption-Shifting Tags (FASTs). We showed that FASTs can be used as alternatives to fluorescent proteins or self-labeling tags to monitor gene expression and protein localization in live cells and organisms. Among the systems developed, we developed pFAST, a chemogenetic reporter with highly tunable spectral and optical properties, allowing investigators to optimize spectral properties at will for multicolor imaging, Förster resonance energy transfer (FRET) experiments and super-resolution microscopy. Protein enegineering allowed us also to generate greenFAST and redFAST, a set of spectrally orthogonal tags well suited for two-color fluorescence microscopy in live cells and multicellular organisms. These orthogonal tags allowed the creation of a two-color cell cycle indicator that enables to delineate cell cycle phases. The quasi-instantaneous fluorescence maturation of greenFAST and redFAST allowed detailed analysis of very short cell cycles, enabling to study cell proliferation in very early stages of embryogenesis with unprecedented temporal resolution. Finally, molecular engineering coupled to directed evolution allowed us to extend the spectral properties to the far-red region. The use of far-red fluorescent tags is particularly important when working with multicellular organisms, due to the lower autofluorescence and the better light penetration in tissues at longer wavelengths. Far-red emitting FAST was shown to be efficient for imaging proteins in multicellular organisms. The modular nature of FASTs allowed us also to develop a new concept of biosensors in which fluorogen binding and thus fluorescence is conditioned by the recognition of a given analyte or recognition event. In particular we developed split fluorescent complementation systems for the monitoring of dynamic protein-protein interactions in live cells. The rapid and reversible complementation of these systems is a unique trait that distinguishes them from split fluorescent proteins that exhibit slow and irreversible complementation because of their chromophore maturation.As the study of complex biological processes requires also tools to acutely control cellular functions, we developed a new method for controlling the activity of cellular processes by controlling protein proximity. Molecular tools enabling to control and observe the proximity of proteins are essential for studying the functional role of physical distance between two proteins. We developed a technology, called CATCHFIRE (Chemically Assisted Tethering of CHimera by Fluorogenic Induced REcognition), a chemically induced proximity technology with intrinsic fluorescence imaging and sensing capabilities.