Biology subjects

Gadella, T. W. J.

Publications and source records attributed to Gadella, T. W. J..

4 recordsLinked to original sources

bioRxiv2017

A FRET based biosensor for measuring Gα13 activation in single cells

Forster Resonance Energy Transfer (FRET) provides a way to directly observe the activation of heterotrimeric G-proteins by G-protein coupled receptors (GPCRs). To this end, FRET based biosensors are made, employing heterotrimeric G-protein subunits tagged with fluorescent proteins. These FRET based biosensors complement existing, indirect, ways to observe GPCR activation. Here we report on the insertion of mTurquoise2 at several sites in the human G13 subunit. Three variants were found to be functional based on i) plasma membrane localization and ii) ability to recruit p115-RhoGEF upon activation of the LPA2 receptor. The tagged G13 subunits were used as FRET donor and combined with cp173Venus fused to the G{gamma}2 subunit as the acceptor. We constructed G13 biosensors by generating a single plasmid that produces G13-mTurquoise2, G{beta}1 and cp173Venus-G{gamma}2. The G13 activation biosensors showed a rapid and robust response when used in primary human endothelial cells that were treated with thrombin, triggering endogenous protease activated receptors (PARs). This response was efficiently inhibited by the RGS domain of p115-RhoGEF and from the biosensor data we inferred that this is due to GAP activity. Finally, we demonstrated that the G13 sensor could be used to dissect heterotrimeric G-protein coupling efficiency in single living cells. We conclude that the G13 biosensor is a valuable tool for live-cell measurements that probe G13 activation.

cell biology↗

bioRxiv2017

Robust and Bright Genetically Encoded Fluorescent Markers for Highlighting Structures and Compartments in Mammalian Cells

To increase our understanding of the inner working of cells, there is a need for specific markers to identify biomolecules, cellular structures and compartments. One type of markers comprises genetically encoded fluorescent probes that are linked with protein domains, peptides and/or signal sequences. These markers are encoded on a plasmid and they allow straightforward, convenient labeling of cultured mammalian cells by introducing the plasmid into the cells. Ideally, the fluorescent marker combines favorable spectroscopic properties (brightness, photostability) with specific labeling of the structure or compartment of interest. Here, we report our ongoing efforts to generate robust and bright genetically encoded fluorescent markers for highlighting structures and compartments in living cells. The plasmids are distributed by addgene: https://www.addgene.org/browse/article/28189953/ O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=71 SRC="FIGDIR/small/160374v2_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@67f3aaorg.highwire.dtl.DTLVardef@ae2235org.highwire.dtl.DTLVardef@47f729org.highwire.dtl.DTLVardef@b3c9d1_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2017

Characterization of a spectrally diverse set of fluorescent proteins as FRET acceptors for mTurquoise2

Genetically encoded Forster Resonance Energy Transfer (FRET) based biosensors report on changes in biochemical states in single living cells. The performance of biosensors depends on their brightness and dynamic range, which are dependent on the characteristics of the fluorescent proteins that are employed. Cyan fluorescent protein (CFP) is frequently combined with yellow fluorescent protein (YFP) as FRET pair in biosensors. However, current YFPs are prone to photobleaching and pH changes. In addition, more efficient acceptors may yield biosensors that have higher contrast. In this study, we evaluated the properties of a diverse set of acceptor fluorescent proteins in combination with the optimized CFP variant mTurquoise2 as the donor. To determine the theoretical performance of acceptors, the Forster radius was determined. The practical performance was determined by measuring FRET efficiency and photostability of tandem fusion proteins in mammalian cells. Our results show that mNeonGreen is the most efficient acceptor for mTurquoise2 and that the photostability is better than SYFP2. The non-fluorescent YFP variant sREACh is an efficient acceptor, which is useful in lifetime-based FRET experiments. Among the orange and red fluorescent proteins, mChery and mScarlet-I are the best performing acceptors. Several new pairs were applied in a multimolecular FRET based sensor for detecting activation of a heterotrimeric G-protein by G-protein coupled receptors. The sensor with mScarlet-I as acceptor and mTurquoise2 as donor shows a higher dynamic range in ratiometric FRET imaging experiments and less variability than with mCherry as acceptor, due to the high quantum yield and efficient maturation of mScarlet-I. Overall, the sensor with mNeonGreen as acceptor and mTurquoise2 as donor showed the highest dynamic range in ratiometric FRET imaging experiments with the G-protein sensor.

cell biology↗

bioRxiv2017

Photorelease of Diacylglycerol Increases the Amplitude and Duration of Protein Kinase C-BetaII Relocation in cyto

Diacylglycerol (DAG) is a lipid second messenger produced by receptor stimulated phospholipase C and is capable of activating several PKC isoforms. Classical PKC isoforms require simultaneous presence of calcium and DAG for activation and relocation to membranes. The aim of this study was to synthesize a photolabile precursor of DAG and examine the effect of an immediate increase of the signaling lipid on PKC relocation. Caged DAG was synthesized using a photoreleasable 7-diethyl-aminocoumarin (DEACM) group. Photolysis was monitored in vitro by an increase in coumarin fluorescence from which an uncaging quantum yield of 1.1% was determined. This quantum yield proved ideal for efficient uncaging at high UV power while allowing localization of the fluorescent compound at low UV power. Taking advantage of the coumarin fluorescence, it was demonstrated that DEACM-DiC8 accumulated inside cells. Effects of DAG photorelease on periodic relocations of PKCbetaII, induced by histamine, UTP or EGF, were studied. Photorelease of DAG in cyto immediately increased the amplitude and duration of relocation events, regardless of the agonist used. Together, the results demonstrate the usefulness of caged DAG for dissecting PKC signaling and suggest that DAG levels are limiting during signaling.

cell biology↗