Biology subjects

Thauvin, M.

Publications and source records attributed to Thauvin, M..

5 recordsLinked to original sources

bioRxiv2021

An early Shh-H2O2 feedback loop controls the progression of the regenerative program during adult zebrafish fin regeneration

Reactive oxygen species (ROS), originally classified as toxic molecules, have attracted increasing interest given their actions in cell signaling. Among these molecules, Hydrogen peroxide (H2O2) is the major ROS produced by cells and acts as a second messenger to modify redox-sensitive proteins or lipids. After amputation, tight spatiotemporal regulation of ROS is required first for wound healing and later to initiate the regenerative program. However, the mechanisms carrying out this sustained ROS production and their integration with signaling pathways are still poorly understood. We focused on the early dialog between H2O2 and Sonic Hedgehog (Shh) during fin regeneration. We demonstrate that H2O2 controls Shh expression and that Shh in turn regulates the H2O2 level via a canonical pathway. Moreover, this tightly controlled feedback loop changes during the successive phases of the regenerative program. Dysregulation of the Hedgehog pathway has been implicated in several developmental syndromes, diabetes and cancer. These data support the existence of a very early feedback loop between Shh and H2O2 that might be more generally involved in various physiological or pathological processes. These new findings pave the way to improve regenerative processes, particularly in vertebrates.

developmental biology↗

bioRxiv2021

Reciprocal regulation of Shh trafficking and H2O2 levels via a noncanonical BOC-Rac1 pathway

Among molecules that bridge environment, cell metabolism, and cell signaling, H2O2 recently appeared as an emerging but central player. Its level depends on cell metabolism and environment and was recently shown to play key roles during embryogenesis, contrasting with its long-established role in disease progression. We decided to explore whether the secreted morphogen Sonic hedgehog (Shh), known to be essential in a variety of biological processes ranging from embryonic development to adult tissue homeostasis and cancers, was part of these interactions. Here, we report that H2O2 levels control key steps of Shh delivery and that physiological in vivo modulation of H2O2 levels changes Shh distribution and tissue patterning. A feedback loop exists in which Shh trafficking controls H2O2 synthesis via a non-canonical BOC-Rac1 pathway, leading to cytoneme growth. Our findings reveal that Shh directly impacts its own distribution, thus providing a molecular explanation for the robustness of morphogenesis to both environmental insults and individual variability.

cell biology↗

bioRxiv2021

Monitoring oxidative inflammatory processes in live cells and tissue with Hypocrates, a genetically encoded biosensor for hypochlorite

Hypochlorous acid, an aggressive oxidant, is important in immune defense against pathogens. The current lack of tools to monitor the dynamics of hypochlorous acid in live cells and tissue hinders a better understanding of inflammatory processes. We engineered a genetically encoded biosensor, Hypocrates, for the visualization of hypochlorous acid. Hypocrates consists of a circularly permuted yellow fluorescent protein integrated into the structure of the transcription repressor NemR from E. coli. We determined sensitivity, selectivity, reaction rates, and the X-ray structure of this ratiometric redox biosensor, and tested the response of Hypocrates in HeLa Kyoto cells at varying hypochlorite concentrations. By combining Hypocrates with the biosensor HyperRed, we visualized the dynamics of hypochlorous acid and hydrogen peroxide in a zebrafish tail fin injury model.

immunology↗

bioRxiv2020

Orthogonal fluorescent chemogenetic reporters for multicolor imaging

Fluorescence microscopy is an indispensable tool in biological research, allowing sub-second and sub-micrometer mapping of molecules or processes inside living cells. Moreover, using spectrally separated fluorophores, one can observe multiple targets simultaneously, leading to a deeper understanding of the dynamic molecular interplays that regulate cell function and fate. Chemogenetic systems, which combine a protein tag and a synthetic fluorophore, provide certain advantages over fluorescent proteins since there is no requirement for chromophore maturation. However, the fluorophore promiscuity of chemogenetic systems renders two-color applications challenging. Here, we present the engineering of a set of spectrally orthogonal fluorogen activating tags based on the Fluorescence Activating and absorption Shifting Tag (FAST), that are compatible with two-color, live cell imaging. The resulting tags, greenFAST and redFAST, demonstrate orthogonality not only in their fluorogen recognition capabilities, but also in their one- and two-photon absorption profiles. A two-color cell cycle sensor based on greenFAST and redFAST is capable of detecting very short, early cell cycles in zebrafish development which had previously been difficult to image. Furthermore, this pair of orthogonal tags can be developed into split complementation systems that are capable of detecting multiple protein-protein interactions by live cell fluorescence microscopy.

molecular biology↗

bioRxiv2020

A far-red fluorescent chemogenetic reporter for in vivo molecular imaging

Far-red emitting fluorescent labels are highly desirable for spectral multiplexing and deep tissue imaging. Here, we describe the generation of frFAST (far-red Fluorescence Activating and absorption Shifting Tag), a 14-kDa monomeric protein that forms a bright far-red fluorescent assembly with (4-hydroxy-3-methoxy-phenyl)allylidene rhodanine (HPAR-3OM). As HPAR-3OM is essentially non-fluorescent in solution and in cells, frFAST can be imaged with high contrast in presence of free HPAR-3OM, which allowed the rapid and efficient imaging of frFAST fusions in live cells, zebrafish embryo/larvae and chicken embryo. Beyond enabling genetic encoding of far-red fluorescence, frFAST allowed the design of a far-red chemogenetic reporter of protein-protein interactions, demonstrating its great potential for the design of innovative far-red emitting biosensors.

molecular biology↗