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19 Publications

Showing 1-10 of 19 results
08/01/20 | Sensing cellular biochemistry with fluorescent chemical–genetic hybrids
Gautier A, Tebo AG
Current Opinion in Chemical Biology. 08/2020;57:58–64. doi: 10.1016/j.cbpa.2020.04.005

Fluorescent biosensors are powerful tools for the detection of biochemical events inside cells with high spatiotemporal resolution. Biosensors based on fluorescent proteins often suffer from issues with photostability and brightness. On the other hand, hybrid, chemical–genetic systems present unique opportunities to combine the strengths of synthetic, organic chemistry with biological macromolecules to generate exquisitely tailored semisynthetic sensors.

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09/25/20 | Integrated structure-function dataset reveals key mechanisms underlying photochromic fluorescent proteins
Zitter ED, Hugelier S, Duwé S, Vandenberg W, Tebo AG, Meervelt LV, Dedecker P
bioRxiv. 09/2020:2020.09.25.313528. doi: 10.1101/2020.09.25.313528

Photochromic fluorescent proteins have become versatile tools in the life sciences, though our understanding of their structure-function relation is limited. Starting from a single scaffold, we have developed a range of 27 photochromic fluorescent proteins that cover a broad range of spectroscopic properties, yet differ only in one or two mutations. We also determined 43 different crystal structures of these mutants. Correlation and principal component analysis of the spectroscopic and structural properties confirmed the complex relationship between structure and spectroscopy, suggesting that the observed variability does not arise from a limited number of mechanisms, but also allowed us to identify consistent trends and to relate these to the spatial organization around the chromophore. We find that particular changes in spectroscopic properties can come about through multiple different underlying mechanisms, of which the polarity of the chromophore environment and hydrogen bonding of the chromophore are key modulators. Furthermore, some spectroscopic parameters, such as the photochromism, appear to be largely determined by a single or a few structural properties, while other parameters, such as the absorption maximum, do not allow a clear identification of a single cause. We also highlight the role of water molecules close to the chromophore in influencing photochromism. We anticipate that our dataset can open opportunities for the development and evaluation of new and existing protein engineering methods.

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04/05/20 | Orthogonal fluorescent chemogenetic reporters for multicolor imaging
Tebo AG, Moeyaert B, Thauvin M, Carlon-Andres I, Böken D, Volovitch M, Padilla-Parra S, Dedecker P, Vriz S, Gautier A
Nature Chemical Biology. 04/2020:1–9. doi: 10.1038/s41589-020-0611-0

Spectrally separated fluorophores allow the observation of multiple targets simultaneously inside living cells, leading to a deeper understanding of the molecular interplay that regulates cell function and fate. Chemogenetic systems combining a tag and a synthetic fluorophore provide certain advantages over fluorescent proteins since there is no requirement for chromophore maturation. 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. This pair of orthogonal tags allowed the creation of a two-color cell cycle sensor capable of detecting very short, early cell cycles in zebrafish development and the development of split complementation systems capable of detecting multiple protein–protein interactions by live-cell fluorescence microscopy. The fluorescent chemogenetic reporters greenFAST and redFAST were engineered by protein engineering. They display orthogonal fluorogen recognition and spectral properties allowing efficient multicolor imaging of proteins in live cells and organisms.

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06/22/20 | A far‐red fluorescent chemogenetic reporter for in vivo molecular imaging
Li C, Tebo AG, Thauvin M, Plamont M, Volovitch M, Morin X, Vriz S, Gautier A
Angewandte Chemie International Edition. 06/2020:. doi: 10.1002/anie.202006576

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.

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07/30/19 | Simple imaging protocol for autofluorescence elimination and optical sectioning in fluorescence endomicroscopy
Zhang R, Chouket R, Tebo AG, Plamont M, Kelemen Z, Gissot L, Faure J, Gautier A, Croquette V, Jullien L, Saux TL
Optica. 07/2019;6:972. doi: 10.1364/optica.6.000972

Fiber-optic epifluorescence imaging with one-photon excitation benefits from its ease of use, cheap light sources, and full-frame acquisition, which enables it for favorable temporal resolution of image acquisition. However, it suffers from a lack of robustness against autofluorescence and light scattering. Moreover, it cannot easily eliminate the out-of-focus background, which generally results in low-contrast images. In order to overcome these limitations, we have implemented fast out-of-phase imaging after optical modulation (Speed OPIOM) for dynamic contrast in fluorescence endomicroscopy. Using a simple and cheap optical-fiber bundle-based endomicroscope integrating modulatable light sources, we first showed that Speed OPIOM provides intrinsic optical sectioning, which restricts the observation of fluorescent labels at targeted positions within a sample. We also demonstrated that this imaging protocol efficiently eliminates the interference of autofluorescence arising from both the fiber bundle and the specimen in several biological samples. Finally, we could perform multiplexed observations of two spectrally similar fluorophores differing by their photoswitching dynamics. Such attractive features of Speed OPIOM in fluorescence endomicroscopy should find applications in bioprocessing, clinical diagnostics, plant observation, and surface imaging.

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06/27/19 | A split fluorescent reporter with rapid and reversible complementation.
Tebo AG, Gautier A
Nature communications. 06/2019;10:2822. doi: 10.1038/s41467-019-10855-0

Interactions between proteins play an essential role in metabolic and signaling pathways, cellular processes and organismal systems. We report the development of splitFAST, a fluorescence complementation system for the visualization of transient protein-protein interactions in living cells. Engineered from the fluorogenic reporter FAST (Fluorescence-Activating and absorption-Shifting Tag), which specifically and reversibly binds fluorogenic hydroxybenzylidene rhodanine (HBR) analogs, splitFAST displays rapid and reversible complementation, allowing the real-time visualization of both the formation and the dissociation of a protein assembly.

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11/29/18 | Macroscale fluorescence imaging against autofluorescence under ambient light
Zhang R, Chouket R, Plamont M, Kelemen Z, Espagne A, Tebo AG, Gautier A, Gissot L, Faure J, Jullien L, Croquette V, Saux TL
Light: Science & Applications. 11/2018:1 – 12. doi: 10.1038/s41377-018-0098-6

Macroscale fluorescence imaging is increasingly used to observe biological samples. However, it may suffer from spectral interferences that originate from ambient light or autofluorescence of the sample or its support. In this manuscript, we built a simple and inexpensive fluorescence macroscope, which has been used to evaluate the performance of Speed OPIOM (Out of Phase Imaging after Optical Modulation), which is a reference-free dynamic contrast protocol, to selectively image reversibly photoswitchable fluorophores as labels against detrimental autofluorescence and ambient light. By tuning the intensity and radial frequency of the modulated illumination to the Speed OPIOM resonance and adopting a phase-sensitive detection scheme that ensures noise rejection, we enhanced the sensitivity and the signal-to-noise ratio for fluorescence detection in blot assays by factors of 50 and 10, respectively, over direct fluorescence observation under constant illumination. Then, we overcame the strong autofluorescence of growth media that are currently used in microbiology and realized multiplexed fluorescence observation of colonies of spectrally similar fluorescent bacteria with a unique configuration of excitation and emission wavelengths. Finally, we easily discriminated fluorescent labels from the autofluorescent and reflective background in labeled leaves, even under the interference of incident light at intensities that are comparable to sunlight. The proposed approach is expected to find multiple applications, from biological assays to outdoor observations, in fluorescence macroimaging.

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09/11/18 | Improved Chemical-Genetic Fluorescent Markers for Live Cell Microscopy
Tebo AG, Pimenta FM, Zhang Y, Gautier A
Biochemistry. 11/2018;57:5648 – 5653. doi: 10.1021/acs.biochem.8b00649

Inducible chemical-genetic fluorescent markers are promising tools for live cell imaging requiring high spatiotemporal resolution and low background fluorescence. The fluorescence-activating and absorption shifting tag (FAST) was recently developed to form fluorescent molecular complexes with a family of small, synthetic fluorogenic chromophores (so-called fluorogens). Here, we use rational design to modify the binding pocket of the protein and screen for improved fluorescence performances with four different fluorogens. The introduction of a single mutation results in improvements in both quantum yield and dissociation constant with nearly all fluorogens tested. Our improved FAST (iFAST) allowed the generation of a tandem iFAST (td-iFAST) that forms green and red fluorescent reporters 1.6-fold and 2-fold brighter than EGFP and mCherry, respectively, while having a comparable size.

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08/28/18 | Fluorogenic Protein‐Based Strategies for Detection, Actuation, and Sensing
Gautier A, Tebo AG
BioEssays. 08/2018;40:1800118. doi: 10.1002/bies.201800118

Fluorescence imaging has become an indispensable tool in cell and molecular biology. GFP‐like fluorescent proteins have revolutionized fluorescence microscopy, giving experimenters exquisite control over the localization and specificity of tagged constructs. However, these systems present certain drawbacks and as such, alternative systems based on a fluorogenic interaction between a chromophore and a protein have been developed. While these systems are initially designed as fluorescent labels, they also present new opportunities for the development of novel labeling and detection strategies. This review focuses on new labeling protocols, actuation methods, and biosensors based on fluorogenic protein systems. This review presents recently developed fluorogenic protein‐based systems made of a protein tag incorporating an external chromophore. Beyond addressing some limitations of classical fluorescent proteins, these unique systems present characteristics than can be used to creatively push the limits of biological imaging, in particular for the development of new labeling protocols, actuation methods and biosensors.

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08/08/18 | Circularly Permuted Fluorogenic Proteins for the Design of Modular Biosensors.
Tebo AG, Pimenta FM, Zoumpoulaki M, Kikuti C, Sirkia H, Plamont M, Houdusse A, Gautier A
ACS Chemical Biology. 09/2018;13:2392 – 2397. doi: 10.1021/acschembio.8b00417

Fluorescent reporters are essential components for the design of optical biosensors that are able to image intracellular analytes in living cells. Herein, we describe the development of circularly permuted variants of Fluorescence-Activating and absorption-Shifting Tag (FAST) and demonstrate their potential as reporting module in biosensors. Circularly permutated FAST (cpFAST) variants allow one to condition the binding and activation of a fluorogenic ligand (and thus fluorescence) to analyte recognition by coupling them with analyte-binding domains. We demonstrated their use for biosensor design by generating multicolor plug-and-play fluorogenic biosensors for imaging the intracellular levels of Ca2+ in living mammalian cells in real time.

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