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A New Generation of FRET Sensors for Robust Measurement of Gαi1, Gαi2 and Gαi3 Activation Kinetics in Single Cells

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      Abstract

      G-protein coupled receptors (GPCRs) can activate a heterotrimeric G-protein complex with subsecond kinetics. Genetically encoded biosensors based on Förster resonance energy transfer (FRET) are ideally suited for the study of such fast signaling events in single living cells. Here we report on the construction and characterization of three FRET biosensors for the measurement of Gα i1, Gα i2 and Gα i3 activation. To enable quantitative long-term imaging of FRET biosensors with high dynamic range, fluorescent proteins with enhanced photophysical properties are required. Therefore, we use the currently brightest and most photostable CFP variant, mTurquoise2, as donor fused to Gα i subunit, and cp173Venus fused to the Gγ 2 subunit as acceptor. The Gα i FRET biosensors constructs are expressed together with Gβ 1 from a single plasmid, providing preferred relative expression levels with reduced variation in mammalian cells. The Gα i FRET sensors showed a robust response to activation of endogenous or over-expressed alpha-2A-adrenergic receptors, which was inhibited by pertussis toxin. Moreover, we observed activation of the Gα i FRET sensor in single cells upon stimulation of several GPCRs, including the LPA 2, M 3 and BK 2 receptor. Furthermore, we show that the sensors are well suited to extract kinetic parameters from fast measurements in the millisecond time range. This new generation of FRET biosensors for Gα i1, Gα i2 and Gα i3 activation will be valuable for live-cell measurements that probe Gα i activation.

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      G-protein-coupled receptors and cancer.

      G-protein-coupled receptors (GPCRs), the largest family of cell-surface molecules involved in signal transmission, have recently emerged as crucial players in tumour growth and metastasis. Malignant cells often hijack the normal physiological functions of GPCRs to survive, proliferate autonomously, evade the immune system, increase their blood supply, invade their surrounding tissues and disseminate to other organs. This Review will address our current understanding of the many roles of GPCRs and their signalling circuitry in tumour progression and metastasis. We will also discuss how interfering with GPCRs might provide unique opportunities for cancer prevention and treatment.
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        Gene splicing and mutagenesis by PCR-driven overlap extension.

        Extension of overlapping gene segments by PCR is a simple, versatile technique for site-directed mutagenesis and gene splicing. Initial PCRs generate overlapping gene segments that are then used as template DNA for another PCR to create a full-length product. Internal primers generate overlapping, complementary 3' ends on the intermediate segments and introduce nucleotide substitutions, insertions or deletions for site-directed mutagenesis, or for gene splicing, encode the nucleotides found at the junction of adjoining gene segments. Overlapping strands of these intermediate products hybridize at this 3' region in a subsequent PCR and are extended to generate the full-length product amplified by flanking primers that can include restriction enzyme sites for inserting the product into an expression vector for cloning purposes. The highly efficient generation of mutant or chimeric genes by this method can easily be accomplished with standard laboratory reagents in approximately 1 week.
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          Expanded dynamic range of fluorescent indicators for Ca(2+) by circularly permuted yellow fluorescent proteins.

          Fluorescence resonance energy transfer (FRET) technology has been used to develop genetically encoded fluorescent indicators for various cellular functions. Although most indicators have cyan- and yellow-emitting fluorescent proteins (CFP and YFP) as FRET donor and acceptor, their poor dynamic range often prevents detection of subtle but significant signals. Here, we optimized the relative orientation of the two chromophores in the Ca(2+) indicator, yellow cameleon (YC), by fusing YFP at different angles. We generated circularly permuted YFPs (cpYFPs) that showed efficient maturation and acid stability. One of the cpYFPs incorporated in YC absorbs a great amount of excited energy from CFP in its Ca(2+)-saturated form, thereby increasing the Ca(2+)-dependent change in the ratio of YFP/CFP by nearly 600%. Both in cultured cells and in the nervous system of transgenic mice, the new YC enables visualization of subcellular Ca(2+) dynamics with better spatial and temporal resolution than before. Our study provides an important guide for the development and improvement of indicators using GFP-based FRET.
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            Author and article information

            Affiliations
            [1 ]Swammerdam Institute for Life Sciences, Section of Molecular Cytology, van Leeuwenhoek Centre for Advanced Microscopy, University of Amsterdam, P.O. Box 94215, NL-1090 GE, Amsterdam, The Netherlands
            [2 ]Bio-Imaging-Center/Rudolf-Virchow-Zentrum and Department of Pharmacology and Toxicology, University of Wuerzburg, Versbacher Strasse 9, 97078, Wuerzburg, Germany
            [3 ]Department of Molecular Cell Biology, Sanquin Research and Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, NL-1066 CX, Amsterdam, the Netherlands
            Boston University School of Medicine, UNITED STATES
            Author notes

            Competing Interests: The authors have declared that no competing interests exist.

            Conceived and designed the experiments: JvU PLH CH JG TWG. Performed the experiments: JvU ADS BS NRR. Analyzed the data: JvU ADS BS NRR CH JG. Wrote the paper: JvU ADS BS NRR PLH CH TWG JG.

            Contributors
            Role: Editor
            Journal
            PLoS One
            PLoS ONE
            plos
            plosone
            PLoS ONE
            Public Library of Science (San Francisco, CA USA )
            1932-6203
            22 January 2016
            2016
            : 11
            : 1
            26799488 4723041 10.1371/journal.pone.0146789 PONE-D-15-42389
            © 2016 van Unen et al

            This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

            Counts
            Figures: 3, Tables: 0, Pages: 14
            Product
            Funding
            The authors have no support or funding to report.
            Categories
            Research Article
            Research and Analysis Methods
            Spectrum Analysis Techniques
            Spectrophotometry
            Fluorophotometry
            Fluorescence Resonance Energy Transfer
            Research and analysis methods
            Biological cultures
            Cell lines
            HeLa cells
            Research and analysis methods
            Biological cultures
            Cell cultures
            Cultured tumor cells
            HeLa cells
            Biology and Life Sciences
            Biochemistry
            Proteins
            Luminescent Proteins
            Yellow Fluorescent Protein
            Biology and Life Sciences
            Biochemistry
            Proteins
            Transmembrane Receptors
            G Protein Coupled Receptors
            Biology and Life Sciences
            Cell Biology
            Signal Transduction
            Transmembrane Receptors
            G Protein Coupled Receptors
            Engineering and Technology
            Equipment
            Detectors
            Biosensors
            Engineering and Technology
            Signal Processing
            Signal Filtering
            Biology and Life Sciences
            Molecular Biology
            Molecular Biology Techniques
            Transfection
            Research and Analysis Methods
            Molecular Biology Techniques
            Transfection
            Biology and Life Sciences
            Molecular Biology
            Molecular Biology Techniques
            Artificial Gene Amplification and Extension
            Polymerase Chain Reaction
            Research and Analysis Methods
            Molecular Biology Techniques
            Artificial Gene Amplification and Extension
            Polymerase Chain Reaction
            Custom metadata
            All relevant data are within the paper and its Supporting Information files.

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