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TRUPATH, an open-source biosensor platform for interrogating the GPCR transducerome.

ABSTRACT: G-protein-coupled receptors (GPCRs) remain major drug targets, despite our incomplete understanding of how they signal through 16 non-visual G-protein signal transducers (collectively named the transducerome) to exert their actions. To address this gap, we have developed an open-source suite of 14 optimized bioluminescence resonance energy transfer (BRET) G??? biosensors (named TRUPATH) to interrogate the transducerome with single pathway resolution in cells. Generated through exhaustive protein engineering and empirical testing, the TRUPATH suite of G??? biosensors includes the first G?15 and G?Gustducin probes. In head-to-head studies, TRUPATH biosensors outperformed first-generation sensors at multiple GPCRs and in different cell lines. Benchmarking studies with TRUPATH biosensors recapitulated previously documented signaling bias and revealed new coupling preferences for prototypic and understudied GPCRs with potential in vivo relevance. To enable a greater understanding of GPCR molecular pharmacology by the scientific community, we have made TRUPATH biosensors easily accessible as a kit through Addgene.

SUBMITTER: Olsen RHJ

PROVIDER: S-EPMC7648517 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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TRUPATH, an open-source biosensor platform for interrogating the GPCR transducerome.

Olsen Reid H J RHJ DiBerto Jeffrey F JF English Justin G JG Glaudin Alexis M AM Krumm Brian E BE Slocum Samuel T ST Che Tao T Gavin Ariana C AC McCorvy John D JD Roth Bryan L BL Strachan Ryan T RT

Nature chemical biology 20200504 8

G-protein-coupled receptors (GPCRs) remain major drug targets, despite our incomplete understanding of how they signal through 16 non-visual G-protein signal transducers (collectively named the transducerome) to exert their actions. To address this gap, we have developed an open-source suite of 14 optimized bioluminescence resonance energy transfer (BRET) Gαβγ biosensors (named TRUPATH) to interrogate the transducerome with single pathway resolution in cells. Generated through exhaustive protein ...[more]

PMID: 32367019

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Project description:BackgroundRemote monitoring of plants using hyperspectral imaging has become an important tool for the study of plant growth, development, and physiology. Many applications are oriented towards use in field environments to enable non-destructive analysis of crop responses due to factors such as drought, nutrient deficiency, and disease, e.g., using tram, drone, or airplane mounted instruments. The field setting introduces a wide range of uncontrolled environmental variables that make validation and interpretation of spectral responses challenging, and as such lab- and greenhouse-deployed systems for plant studies and phenotyping are of increasing interest. In this study, we have designed and developed an open-source, hyperspectral reflectance-based imaging system for lab-based plant experiments: the HyperScanner. The reliability and accuracy of HyperScanner were validated using drought and salt stress experiments with Arabidopsis thaliana.ResultsA robust, scalable, and reliable system was created. The system was built using open-sourced parts, and all custom parts, operational methods, and data have been made publicly available in order to maintain the open-source aim of HyperScanner. The gathered reflectance images showed changes in narrowband red and infrared reflectance spectra for each of the stress tests that was evident prior to other visual physiological responses and exhibited congruence with measurements using full-range contact spectrometers.ConclusionsHyperScanner offers the potential for reliable and inexpensive laboratory hyperspectral imaging systems. HyperScanner was able to quickly collect accurate reflectance curves on a variety of plant stress experiments. The resulting images showed spectral differences in plants shortly after application of a treatment but before visual manifestation. HyperScanner increases the capacity for spectroscopic and imaging-based analytical tools by providing more access to hyperspectral analyses in the laboratory setting.

Dataset Information

TRUPATH, an open-source biosensor platform for interrogating the GPCR transducerome.

Publications

TRUPATH, an open-source biosensor platform for interrogating the GPCR transducerome.

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