G-Protein-Dependent Cell Surface Dynamics of the Human Serotonin1AReceptor Tagged to Yellow Fluorescent Protein†

The RGS (regulators of G-protein signalling) protein family sharpen signalling kinetics through heterotrimeric G-proteins by enhancing the GTPase activity of the G-protein α subunit. Paradoxically, they also accelerate receptor-stimulated activation. We investigated this paradox using the cloned G-protein gated K+ channel as a reporter of the G-protein cycle, and FRET (fluorescence resonance energy transfer) between cyan and yellow fluorescent protein tagged proteins to detect physical interactions. Our results with the neuronal protein, RGS8, show that the enhancement of activation kinetics is a variable phenomenon determined by receptor type, G-protein isoform and RGS8 expression levels. In contrast, deactivation was consistently accelerated after removal of agonist. FRET microscopy revealed a stable physical interaction between RGS8-yellow fluorescent protein and Go αA-cyan fluorescent protein that occurred in the presence and absence of receptor activation and was not competed away by Gβγ overexpression. FRET was also seen between RGS8 and Gγ, demonstrating that RGS8 binds to the heterotrimeric G-protein as well as G-protein α subunit-GTP and the transition complex. We propose a novel model for the action of RGS proteins on the G-protein cycle involving participation of the RGS in the ternary complex: for certain combinations of agonist, receptor and G-protein, RGS8 expression improves upon the ‘kinetic efficacy’ of G-protein activation.

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Apical membrane targeting and trafficking of the human proton-coupled transporter in polarized epithelia

AJP Cell Physiology ◽

10.1152/ajpcell.00468.2007 ◽

2008 ◽

Vol 294 (1) ◽

pp. C233-C240 ◽

Cited By ~ 41

Author(s):

Veedamali S. Subramanian ◽

Jonathan S. Marchant ◽

Hamid M. Said

Keyword(s):

Cell Surface ◽

Structure Function ◽

Fluorescent Protein ◽

Apical Membrane ◽

Yellow Fluorescent Protein ◽

Apical Cell ◽

Membrane Targeting ◽

Acid Uptake ◽

Conserved Sequence ◽

Polarized Cells

The human proton-coupled folate transporter (hPCFT) is a recently discovered intestinal transporter involved in folate uptake in epithelia (and possibly other cells). Little is currently known about the structure-function relationship of the different domains of this transporter, particularly which regions are important for substrate transport as well as targeting of the transporter to the apical cell surface of polarized cells. Here we have investigated the role of the COOH-terminal domain and a well-conserved sequence separating transmembrane (TM) domains TM2 and TM3 (DXXGRR; amino acids 109–114) speculated by others to be important for transport function. Using live cell imaging approaches, we show that 1) an hPCFT-yellow fluorescent protein construct is functionally expressed at the apical membrane domain and is localized differentially to the human reduced folate carrier; 2) the predicted cytoplasmic COOH-terminal region of hPCFT is not essential for apical targeting or transporter functionality; 3) mutations that ablate a consensus β-turn sequence separating predicted TM2 and TM3 abolished apical [3H]folic acid uptake as a consequence of endoplasmic reticulum retention of mutant, likely misfolded, transporters; and 4) cell surface delivery of hPCFT is disrupted by microtubule depolymerization or by overexpression of the dynactin complex dynamitin (p50). For the first time, our data present information regarding structure-function and membrane targeting of the hPCFT polypeptide, as well as the mechanisms that control its steady-state expression in polarized cells.

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A Model to Study Platelet Integrin Variants Using Transfected Human Embryonic Kidney-293 Cells Expressing Fluorescent αIIbβ3-Fusion Proteins

Blood ◽

10.1182/blood.v112.11.5364.5364 ◽

2008 ◽

Vol 112 (11) ◽

pp. 5364-5364

Author(s):

Volker Rainer Stoldt ◽

Abdelouahid El Khattouti ◽

Rudiger E. Scharf

Keyword(s):

Fusion Protein ◽

G Protein ◽

Fluorescent Protein ◽

Yellow Fluorescent Protein ◽

Laser Scanning Microscopy ◽

Hek293 Cells ◽

Confocal Laser ◽

Chimeric Antibody ◽

Human Embryonic Kidney ◽

G Protein Coupled

Abstract The HPA-1 polymorphism of platelet integrin αIIbβ3 (GPIIb-IIIa) arises from a thymidine to cytosine transition in position 1565 of the ITGB3 gene. This transition leads to an amino acid exchange at residue 33 of the mature β3 subunit. The resulting isoforms are HPA-1a (Leu33) or HPA-1b (Pro33). We have shown that the HPA-1b variant of αIIbβ3 is associated with premature manifestation of myocardial infarction in patients suffering from coronary artery disease (Zotz et al. J Thromb Haemost 2005). This observation has lead to the hypothesis that the HPA-1b variant of αIIbβ3 may increase platelet thrombogenicity. Recently, we have also demonstrated that HPA-1b/1b platelets adhering onto fibrinogen are more resistant than HPA-1a/1a platelets when exposed to arterial shear rates of 1000 to 1500 sec-1 (Loncar et al. Thromb J 2007). To explore the molecular nature of the postulated prothrombotic phenotype of HPA-1b in further detail, we have now overexpressed the yellow fluorescent protein (YFP) or the cyan fluorescent protein (CFP) fused to the cytoplasmic tails of the αIIb or β3 subunit of both αIIbβ3 variants in human embryonic kidney-293 (HEK293) cells. Clones were screened for their cyan and yellow fluorescence. Ten positive clones of each αIIbβ3 variant were detected and characterized by Western blotting identifying the 140 kD αIIb-CFP fusion protein and the 113 kD β3-YFP fusion protein with appropriate antibodies directed against the αIIb subunit, the β3 subunit or the fluorescent proteins. Stable HEK293 clones expressing the HPA-1 receptor isoforms on the cell surface were functionally analyzed by flow cytometry (Becton Dickinson), confocal laser scanning microscopy (Zeiss), and a fluorescence imager (Thermo). We used fluorophore (PE)-conjugated complex-specific (PM6/248) and activation-dependent (PAC-1) antibodies and fluorescently tagged fibrinogen (Alexa647- Fg) in combination with phorbol-12-myristate-13-acetate (PMA) or organic acid (1-stearoyl-2-arachidonoyl-sn-glycerol, SAG). Corresponding controls were performed with the chimeric antibody abciximab (ReoPro) to block fibrinogen binding to αIIbβ3 or with pertussis toxin (PTX) to inhibit G-protein-coupled inside-out signal transduction. Functional integrity of the integrin variants (HPA-1a and HPA-1b) was demonstrated by intact activation through G-protein-coupled receptors with SAG and by specific binding of Alexa647 fibrinogen to αIIbβ3 indicating proper insertion of the receptor complex into the plasma membrane of transfected cells. In the presence of PTX or abciximab, activation and ligand binding function of αIIbβ3 were completely (>95%) inhibited in both isoforms of HPA-1. Cytoplasmic conformational changes upon integrin activation using either PMA or SAG were followed by fluorescence resonance energy transfer (FRET) and quantified by FRET signal disappearance due to allosteric changes of the cytoplasmic domains. Stimulation with PMA and SAG caused FRET signal disappearance to same extent in each HPA-1 variant. However, the dynamics of signal disappearance appeared to be faster in the HPA-1b than in the HPA-1a variant of the cell clones studied so far. This observation corresponds to the prothrombotic phenotype of HPA-1b. In conclusion, our results demonstrate that we have generated a cellular model which can be useful to study molecular properties of αIIbβ3 variants and to explore the nature of the prothrombotic HPA-1b phenotype in further detail.

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Identification of the roles of conserved charged residues in the extracellular domain of an epithelial sodium channel (ENaC) subunit by alanine mutagenesis

AJP Renal Physiology ◽

10.1152/ajprenal.00648.2010 ◽

2011 ◽

Vol 300 (4) ◽

pp. F887-F897 ◽

Cited By ~ 19

Author(s):

Oded Edelheit ◽

Israel Hanukoglu ◽

Nathan Dascal ◽

Aaron Hanukoglu

Keyword(s):

Cell Surface ◽

Fluorescent Protein ◽

Yellow Fluorescent Protein ◽

Surface Expression ◽

Cell Surface Expression ◽

Xenopus Laevis Oocytes ◽

Epithelial Sodium Channels ◽

Charged Residues ◽

Inhibition Control ◽

Two Parameters

Epithelial sodium channels (ENaC) are composed of three homologous subunits whose extracellular domains (ECD) form a funnel that directs ions from the lumen into the pore of ENaC. To examine the roles of conserved charged residues (Asp, Glu, Arg, and Lys) on ECD, we mutated 16 residues in human α-ENaC to alanine. The modified cRNAs were expressed in Xenopus laevis oocytes together with wild-type β- and γ-ENaC. The effect of each mutation was examined on three parameters: amiloride-sensitive Na+ conductance (assayed by the two-electrode voltage-clamp method), Na+-dependent self-inhibition of ENaC, and oocyte cell surface expression of ENaC (quantitated by confocal microscopy of yellow fluorescent protein linked to γ-ENaC). Mutation of 13 of 16 residues reduced the ENaC Na+ conductance (to 40–80% of WT). Mutation of only six residues showed a significant effect on the Na+ self-inhibition time constant (τ). All 16 mutants showed a strong correlation between ENaC activity and oocyte surface expression ( r = 0.62). Exclusion of four mutants showing the greatest effect on self-inhibition kinetics (Glu250 and Arg350 with τ = ∼30% of WT, and Asp393 and Glu530 with τ = ∼170% of WT) increased the correlation to r = 0.87. In the ASIC1 homotrimeric model, the homologs of α-ENaC Asp400 and Asp446 are exposed on the protein surface far from the other two chains. The mutations of these two residues showed the strongest effect on cell surface expression but had no effect on self-inhibition. Control mutations to a homologous charged residue (e.g., Asp to Glu) did not significantly affect ENaC activity. Changes in the two parameters, Na+ self-inhibition and oocyte surface expression level, accounted for the magnitude of reduction in ENaC activity as a result of the mutation to Ala. These results establish that while some conserved charged residues are part of the structure responsible for Na+ self-inhibition, most are essential for transport to the oocyte cell surface.

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Correlation of Autophagosome Formation with Degradation and Endocytosis Arabidopsis Regulator of G-Protein Signaling (RGS1) through ATG8a

International Journal of Molecular Sciences ◽

10.3390/ijms20174190 ◽

2019 ◽

Vol 20 (17) ◽

pp. 4190 ◽

Cited By ~ 1

Author(s):

Yue Jiao ◽

Miroslav Srba ◽

Jingchun Wang ◽

Wenli Chen

Keyword(s):

G Protein ◽

Fluorescent Protein ◽

Yellow Fluorescent Protein ◽

Autophagic Flux ◽

G Protein Signaling ◽

Protein Signaling ◽

In Planta ◽

Autophagosome Formation ◽

Root Cells ◽

Ubiquitin Proteasome

Damaged or unwanted cellular proteins are degraded by either autophagy or the ubiquitin/proteasome pathway. In Arabidopsis thaliana, sensing of D-glucose is achieved by the heterotrimeric G protein complex and regulator of G-protein signaling 1 (AtRGS1). Here, we showed that starvation increases proteasome-independent AtRGS1 degradation, and it is correlated with increased autophagic flux. RGS1 promoted the production of autophagosomes and autophagic flux; RGS1-yellow fluorescent protein (YFP) was surrounded by vacuolar dye FM4-64 (red fluorescence). RGS1 and autophagosomes co-localized in the root cells of Arabidopsis and BY-2 cells. We demonstrated that the autophagosome marker ATG8a interacts with AtRGS1 and its shorter form with truncation of the seven transmembrane and RGS1 domains in planta. Altogether, our data indicated the correlation of autophagosome formation with degradation and endocytosis of AtRGS1 through ATG8a.

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Rapid three-dimensional imaging of individual insulin release events by Nipkow disc confocal microscopy

Biochemical Society Transactions ◽

10.1042/bst0340675 ◽

2006 ◽

Vol 34 (5) ◽

pp. 675-678 ◽

Cited By ~ 14

Author(s):

G.A. Rutter ◽

M.K. Loder ◽

M.A. Ravier

Keyword(s):

Confocal Microscopy ◽

Cell Surface ◽

Spatial Organization ◽

Fluorescent Protein ◽

Yellow Fluorescent Protein ◽

Three Dimensional ◽

Confocal Imaging ◽

Β Cells ◽

Large Dense Core Vesicles ◽

Cell Depolarization

Minute-to-minute control of the release of insulin by pancreatic β-cells in response to glucose or other stimuli requires the precise delivery of large dense-core vesicles to the plasma membrane and regulated exocytosis. At present, the precise spatial organization at the cell surface and the nature of these events (‘transient’ versus ‘full fusion’) are debated. In order to monitor secretory events simultaneously over most of the surface of clusters of single MIN6 β-cells, we have expressed recombinant neuropeptide Y-Venus (an enhanced and vesicle-targeted form of yellow fluorescent protein) as an insulin surrogate. Individual exocytotic events were monitored using Nipkow spinning disc confocal microscopy, with acquisition of a three-dimensional complete image (eight to twelve confocal slices) in <1 s, in response to cell depolarization. Corroborating earlier studies using TIRF (total internal reflection fluorescence) microscopy, this approach indicates that events occur with roughly equal probability over the entire cell surface, with only minimal clustering in individual areas, and provides no evidence for multiple events at the same site. Nipkow disc confocal imaging may thus provide a useful tool to determine whether event types occur at different sites at the cell surface and to explore the role of endocytic proteins including dynamin-1 and -2 in terminating individual exocytotic events.

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

10.1101/156448 ◽

2017 ◽

Author(s):

Marieke Mastop ◽

Daphne S. Bindels ◽

Nathan C. Shaner ◽

Marten Postma ◽

Theodorus W. J. Gadella ◽

...

Keyword(s):

G Protein ◽

Mammalian Cells ◽

Fluorescent Protein ◽

Dynamic Range ◽

Fluorescent Proteins ◽

Yellow Fluorescent Protein ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Tandem Fusion ◽

Red Fluorescent Proteins

AbstractGenetically encoded Förster 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 Förster 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.

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A molecular rack and pinion actuates a cell-surface adhesin and enables bacterial gliding motility

10.1101/267583 ◽

2018 ◽

Author(s):

Abhishek Shrivastava ◽

Howard C. Berg

Keyword(s):

Cell Surface ◽

Fluorescent Protein ◽

Rotation Axis ◽

Yellow Fluorescent Protein ◽

Gliding Motility ◽

Flavobacterium Johnsoniae ◽

A Cell ◽

Solid Substratum ◽

Gliding Bacterium ◽

Rotary Motor

AbstractThe mechanism for bacterial gliding is not understood. The gliding bacteriumFlavobacterium johnsoniaeis known to have an adhesin, SprB, that moves along the cell surface on a spiral track. When cells are sheared by passage of a suspension through thin tubing, they stop gliding but can be tethered by addition of an anti-SprB antibody. Tethered cells spin about 3 Hz. We labeled the Type 9 secretion system (T9SS) with a yellow-fluorescent-protein (YFP) fusion of GldL. When labeled cells were tethered, a yellow fluorescent spot was found near the rotation axis, which shows that the motor that drives the rotation localizes with the T9SS. The spiral track was labeled by following the motion of Cy3 attached to SprB via an antibody. The distance between the rotation axis and the track was determined by a measurement involving both labels, YFP and Cy3, yielding 90 nm. If a rotary motor spins a pinion of radius 90 nm 3 Hz, a spot on its periphery will move 1.5 μm/s, the speed at which cells glide. We suggest that the pinion drives a flexible tread that carries SprB along a track fixed to the cell surface. Cells glide when such an adhesin adheres to the solid substratum.

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Fusion of Constitutive Membrane Traffic with the Cell Surface Observed by Evanescent Wave Microscopy

The Journal of Cell Biology ◽

10.1083/jcb.149.1.33 ◽

2000 ◽

Vol 149 (1) ◽

pp. 33-40 ◽

Cited By ~ 111

Author(s):

Derek Toomre ◽

Jürgen A. Steyer ◽

Patrick Keller ◽

Wolfhard Almers ◽

Kai Simons

Keyword(s):

Cell Surface ◽

Vesicular Stomatitis Virus ◽

Evanescent Wave ◽

Total Internal Reflection ◽

Fluorescent Protein ◽

Yellow Fluorescent Protein ◽

Wide Field ◽

Protein Fusion ◽

Vesicular Stomatitis Virus Glycoprotein ◽

Vesicular Stomatitis

Monitoring the fusion of constitutive traffic with the plasma membrane has remained largely elusive. Ideally, fusion would be monitored with high spatial and temporal resolution. Recently, total internal reflection (TIR) microscopy was used to study regulated exocytosis of fluorescently labeled chromaffin granules. In this technique, only the bottom cellular surface is illuminated by an exponentially decaying evanescent wave of light. We have used a prism type TIR setup with a penetration depth of ∼50 nm to monitor constitutive fusion of vesicular stomatitis virus glycoprotein tagged with the yellow fluorescent protein. Fusion of single transport containers (TCs) was clearly observed and gave a distinct analytical signature. TCs approached the membrane, appeared to dock, and later rapidly fuse, releasing a bright fluorescent cloud into the membrane. Observation and analysis provided insight about their dynamics, kinetics, and position before and during fusion. Combining TIR and wide-field microscopy allowed us to follow constitutive cargo from the Golgi complex to the cell surface. Our observations include the following: (1) local restrained movement of TCs near the membrane before fusion; (2) apparent anchoring near the cell surface; (3) heterogeneously sized TCs fused either completely; or (4) occasionally larger tubular-vesicular TCs partially fused at their tips.

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