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Pushing the frontiers of biological imaging with genetically encoded fluorescence switches

Periodic Reporting for period 4 - FLUOSWITCH (Pushing the frontiers of biological imaging with genetically encoded fluorescence switches)

Reporting period: 2021-03-01 to 2022-03-31

Cells and organisms are complex machines driven by a set of dynamic biological events tightly orchestrated in space and time. Our understanding of their inner workings is intricately related to our ability to observe how their constituents organize and interact. Despite spectacular developments in biological imaging and probe design, many biologically relevant molecules and processes remain invisible. This project aims at inventing new molecular tools for observing biomolecules and dynamic biochemical events in live cells and tissues with high spatial and temporal resolutions. These tools can allow biologists to address questions ranging from fundamental mechanisms, to the causes of disease and the development of novel therapeutics.
The FLUOSWITCH project aims at developing next-generation fluorescent reporters for multiplexed and high-resolution imaging. Our tools rely on genetically encodable protein tags that bind reversibly fluorogenic dyes (so-called fluorogens) and activate their fluorescence. Fluorogenic dyes are chromophores that are non-fluorescent by their own but give strongly fluorescent complex when bound to a complementary receptor, allowing high contrast imaging. We applied this strategy to create a collection of new fluorescent reporters called FASTs (Fluorescence-Activating and absorption-Shifting Tags), and demonstrated their use for various applications in biological imaging (e.g; Multicolor imaging, Super-resolution imaging, selective cell-surface protein labeling). These developments allowed us furthermore to design tools for imaging biologically relevant molecules and events for which there is currently a lack of observation tools, such as small analytes or protein-protein interactions. Indeed, FASTs provide an attractive alternative to fluorescent proteins for the design of biosensors, because they display many of the same advantages, while the binding of the fluorogenic chromophore, and thus the fluorescence, can be conditioned to the recognition of a given analyte or to a given cellular signal.
By the end of the project, we expect to have a collection of FASTs covering the entire visible spectrum in terms of emission wavelength, using fluorogens with different cell-uptake ability for selective intracellular or extracellular labeling. The second part of the project focuses on the design of: (i) biosensors for imaging various analytes, including neurotransmitters, ions, second messengers and endogenous proteins, (ii) biosensors for detecting key metabolites in biological samples for diagnostic applications, and (iii) the creation of probes for the live-cell observation of specific RNA molecules and genomic DNA loci in living cells. In a last part of the project not yet started, we will focus on the design of signal integrators for cell circuit mapping. The identification of circuits of active cells is essential in understanding complex behaviors. To do so, we propose to design genetically encoded molecular tools acting as integrators to permanently label transiently activated cells for post hoc analysis of the entire system. Our signal integrators will be composed of a genetically encoded sensing unit that can convert genetically encoded non-fluorescent precursors into a fluorescent species in presence of a given signal, but is otherwise inactive in its absence. Repetitive activation of a given subset of cells will thus lead to specific accumulation of fluorophores in activated cells. This fluorophore accumulation will lead to signal amplification and thus high contrast, facilitating post hoc detection of active cell circuits.