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      A New Generation of FRET Sensors for Robust Measurement of Gα i1, Gα i2 and Gα i3 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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          Most cited references 44

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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

                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
                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.

                Article
                PONE-D-15-42389
                10.1371/journal.pone.0146789
                4723041
                26799488
                © 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.

                Page count
                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.

                Uncategorized

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