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Small fluorescence-activating and absorption-shifting tag for tunable protein imaging in vivo.

ABSTRACT: This paper presents Yellow Fluorescence-Activating and absorption-Shifting Tag (Y-FAST), a small monomeric protein tag, half as large as the green fluorescent protein, enabling fluorescent labeling of proteins in a reversible and specific manner through the reversible binding and activation of a cell-permeant and nontoxic fluorogenic ligand (a so-called fluorogen). A unique fluorogen activation mechanism based on two spectroscopic changes, increase of fluorescence quantum yield and absorption red shift, provides high labeling selectivity. Y-FAST was engineered from the 14-kDa photoactive yellow protein by directed evolution using yeast display and fluorescence-activated cell sorting. Y-FAST is as bright as common fluorescent proteins, exhibits good photostability, and allows the efficient labeling of proteins in various organelles and hosts. Upon fluorogen binding, fluorescence appears instantaneously, allowing monitoring of rapid processes in near real time. Y-FAST distinguishes itself from other tagging systems because the fluorogen binding is highly dynamic and fully reversible, which enables rapid labeling and unlabeling of proteins by addition and withdrawal of the fluorogen, opening new exciting prospects for the development of multiplexing imaging protocols based on sequential labeling.

SUBMITTER: Plamont MA

PROVIDER: S-EPMC4725535 | biostudies-other | 2016 Jan

REPOSITORIES: biostudies-other

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Small fluorescence-activating and absorption-shifting tag for tunable protein imaging in vivo.

Plamont Marie-Aude MA Billon-Denis Emmanuelle E Maurin Sylvie S Gauron Carole C Pimenta Frederico M FM Specht Christian G CG Shi Jian J Quérard Jérôme J Pan Buyan B Rossignol Julien J Moncoq Karine K Morellet Nelly N Volovitch Michel M Volovitch Michel M Lescop Ewen E Chen Yong Y Triller Antoine A Vriz Sophie S Le Saux Thomas T Jullien Ludovic L Gautier Arnaud A

Proceedings of the National Academy of Sciences of the United States of America 20151228 3

This paper presents Yellow Fluorescence-Activating and absorption-Shifting Tag (Y-FAST), a small monomeric protein tag, half as large as the green fluorescent protein, enabling fluorescent labeling of proteins in a reversible and specific manner through the reversible binding and activation of a cell-permeant and nontoxic fluorogenic ligand (a so-called fluorogen). A unique fluorogen activation mechanism based on two spectroscopic changes, increase of fluorescence quantum yield and absorption re ...[more]

PMID: 26711992

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Project description:Live-cell fluorescence imaging of methanogenic archaea has been limited due to the strictly anoxic conditions required for growth and issues with autofluorescence associated with electron carriers in central metabolism. Here, we show that the fluorescence-activating and absorption-shifting tag (FAST) complexed with the fluorogenic ligand 4-hydroxy-3-methylbenzylidene-rhodanine (HMBR) overcomes these issues and displays robust fluorescence in Methanococcus maripaludis. We also describe a mechanism to visualize cells under anoxic conditions using a fluorescence microscope. Derivatives of FAST were successfully applied for protein abundance analysis, subcellular localization analysis, and determination of protein-protein interactions. FAST fusions to both formate dehydrogenase (Fdh) and F420-reducing hydrogenase (Fru) displayed increased fluorescence in cells grown on formate-containing medium, consistent with previous studies suggesting the increased abundance of these proteins in the absence of H2. Additionally, FAST fusions to both Fru and the ATPase associated with the archaellum (FlaI) showed a membrane localization in single cells observed using anoxic fluorescence microscopy. Finally, a split reporter translationally fused to the alpha and beta subunits of Fdh reconstituted a functionally fluorescent molecule in vivo via bimolecular fluorescence complementation. Together, these observations demonstrate the utility of FAST as a tool for studying members of the methanogenic archaea. IMPORTANCE Methanogenic archaea are important members of anaerobic microbial communities where they catalyze essential reactions in the degradation of organic matter. Developing additional tools for studying the cell biology of these organisms is essential to understanding them at a mechanistic level. Here, we show that FAST, in combination with the fluorogenic ligand HMBR, can be used to monitor protein dynamics in live cells of M. maripaludis. The application of FAST holds promise for future studies focused on the metabolism and physiology of methanogenic archaea.

Project description:The human microbiome is predominantly composed of facultative and obligate anaerobic bacteria that live in hypoxic/anoxic polymicrobial biofilm communities. Given the oxidative sensitivity of large fractions of the human microbiota, green fluorescent protein (GFP) and related genetically-encoded fluorophores only offer limited utility for live cell imaging due the oxygen requirement for chromophore maturation. Consequently, new fluorescent imaging modalities are needed to study polymicrobial interactions and microbiome-host interactions within anaerobic environments. The fluorescence-activating and absorption shifting tag (FAST) is a rapidly developing genetically-encoded fluorescent imaging technology that exhibits tremendous potential to address this need. In the FAST system, fluorescence only occurs when the FAST protein is complexed with one of a suite of cognate small molecule fluorogens. To expand the utility of FAST imaging, we sought to develop a modular platform (Click-FAST) to democratize fluorogen engineering for personalized use cases. Using Click-FAST, investigators can quickly and affordably sample a vast chemical space of compounds, potentially imparting a broad range of desired functionalities to the parental fluorogen. In this work, we demonstrate the utility of the Click-FAST platform using a novel fluorogen, PLBlaze-alkyne, which incorporates the widely available small molecule ethylvanillin as the hydroxybenzylidine head group. Different azido reagents were clicked onto PLBlaze-alkyne and shown to impart useful characteristics to the fluorogen, such as selective bacterial labeling in mixed populations as well as fluorescent signal enhancement. Conjugation of an 80 Å PEG molecule to PLBlaze-alkyne illustrates the broad size range of functional fluorogen chimeras that can be employed. This PEGylated fluorogen also functions as an exquisitely selective membrane permeability marker capable of outperforming propidium iodide as a fluorescent marker of cell viability.

Project description:A big challenge in the clinical use of Cerenkov luminescence (CL) imaging is its low signal intensity, which is several orders of magnitude below ambient light. Consequently, highly sensitive cameras, sufficient shielding from background light, and long acquisition times are required. To alleviate this problem, we hypothesized a strategy to convert the weak CL signal into a stronger fluorescence signal by using CL-activated formation of nitrenes from azides to locally fix a fluorescent probe in tissue by the formation of a covalent bond. CL-activated drug delivery was also evaluated using the same azide chemistry. The specific delivery of the CL-activated drug to cancer cells could reduce systemic toxicity, which is a limitation in chemotherapy. Methods: A cyanine-class near-infrared fluorescent dye, Cy7, and doxorubicin were synthetically attached to polyfluorinated aryl azide to form Cy7 azide and DOX azide, respectively. Fibrosarcoma cells were incubated with 18F-FDG and exposed to Cy7 azide with subsequent fluorescence imaging. For CL-activated tagging in vivo, tumor-bearing mice were injected first with 90Y-DOTA-RGD, targeting ?v?3 integrins, and then with the Cy7 azide. Fluorescence signal was imaged over time. Breast cancer cells were incubated with DOX azide and 68Ga, after which cell viability was quantified using an assay. Results: CL photoactivation of Cy7 azide in vitro showed significantly higher fluorescence signal from 18F-FDG-treated than untreated cells. In vivo, CL photoactivation could be shown by using the tumor-specific, integrin-targeting 90Y-DOTA-RGD and the localized activation of Cy7 azide. Here, localized CL-induced fluorescence was detected in the tumors and remained significantly higher over several days than in tumors without CL. We also established as a next step CL-activated drug delivery of DOX azide by showing significantly decreasing cell viability of breast cancer cells in a CL dose-dependent manner in vitro using CL photoactivation of DOX azide. Conclusion: We were able to develop a CL-activated "sticky tag" that converts the low CL signal into a stable and long-lasting, highly intense fluorescence signal. This fluorescent footprint of the radioactive signal might be clinically used for intraoperative surgery. The CL-targeted drug delivery strategy may potentially be used for dual-step targeted therapy.

Dataset Information

Small fluorescence-activating and absorption-shifting tag for tunable protein imaging in vivo.

Publications

Small fluorescence-activating and absorption-shifting tag for tunable protein imaging in vivo.

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