scholarly journals pHuji, a pH-sensitive red fluorescent protein for imaging of exo- and endocytosis

2014 ◽  
Vol 207 (3) ◽  
pp. 419-432 ◽  
Author(s):  
Yi Shen ◽  
Morgane Rosendale ◽  
Robert E. Campbell ◽  
David Perrais
Keyword(s):  
Transferrin Receptor ◽  
Adrenergic Receptor ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Ph Sensitive ◽  
Vesicle Formation ◽  
Red Fluorescent Protein ◽  
Endocytic Vesicle ◽  
Β2 Adrenergic Receptor ◽  
Green Fluorescent

Fluorescent proteins with pH-sensitive fluorescence are valuable tools for the imaging of exocytosis and endocytosis. The Aequorea green fluorescent protein mutant superecliptic pHluorin (SEP) is particularly well suited to these applications. Here we describe pHuji, a red fluorescent protein with a pH sensitivity that approaches that of SEP, making it amenable for detection of single exocytosis and endocytosis events. To demonstrate the utility of the pHuji plus SEP pair, we perform simultaneous two-color imaging of clathrin-mediated internalization of both the transferrin receptor and the β2 adrenergic receptor. These experiments reveal that the two receptors are differentially sorted at the time of endocytic vesicle formation.

scholarly journals Probing chemotaxis activity inEscherichia coliusing fluorescent protein fusions

2018 ◽  
Author(s):  
Clémence Roggo ◽  
Jan Roelof van der Meer
Keyword(s):  
Escherichia Coli ◽  
Protein Interactions ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Bacterial Chemotaxis ◽  
Ph Sensitive ◽  
Flagellar Motors ◽  
Receptor Interactions ◽  
Split Egfp ◽  
Green Fluorescent

ABSTRACTChemotaxis is based on ligand-receptor interactions that are transmitted via protein-protein interactions to the flagellar motors. Ligand-receptor interactions in chemotaxis can be deployed for the development of rapid biosensor assays, but there is no consensus as to what the best readout of such assays would have to be. Here we explore two potential fluorescent readouts of chemotactically activeEscherichia colicells. In the first, we probed interactions between the chemotaxis signaling proteins CheY and CheZ by fusing them individually with non-fluorescent parts of a ‘split’-Green Fluorescent Protein. Wild-type chemotactic cells but not mutants lacking the CheA kinase produced distinguishable fluorescence foci, two-thirds of which localize at the cell poles with the chemoreceptors and one-third at motor complexes. Cells expressing fusion proteins only were attracted to serine sources, demonstrating measurable functional interactions between CheY~P and CheZ. Fluorescent foci based on stable split-eGFP displayed small fluctuations in cells exposed to attractant or repellent, but those based on an unstable ASV-tagged eGFP showed a higher dynamic behaviour both in the foci intensity changes and the number of foci per cell. For the second readout, we expressed the pH-sensitive fluorophore pHluorin in the cyto- and periplasm of chemotactically activeE. coli. Calibrations of pHluorin fluorescence as a function of pH demonstrated that cells accumulating near a chemo-attractant temporally increase cytoplasmic pH while decreasing periplasmic pH. Both readouts thus show promise as proxies for chemotaxis activity, but will have to be further optimized in order to deliver practical biosensor assays.IMPORTANCEBacterial chemotaxis may be deployed for future biosensing purposes with the advantages of its chemoreceptor ligand-specificity and its minute-scale response time. On the downside, chemotaxis is ephemeral and more difficult to quantitatively read out than, e.g., reporter gene expression. It is thus important to investigate different alternative ways to interrogate chemotactic response of cells. Here we gauge the possibilities to measure dynamic response in theEscherichia colichemotaxis pathway resulting from phosphorylated CheY-CheZ interactions by using (unstable) split-fluorescent proteins. We further test whether pH differences between cyto- and periplasm as a result of chemotactic activity can be measured with help of pH-sensitive fluorescent proteins. Our results show that both approaches conceptually function, but will need further improvement in terms of detection and assay types to be practical for biosensing.

scholarly journals Nitrosative stress uncovers potent β2-adrenergic receptor-linked vasodilation further enhanced by blockade of clathrin endosome formation

2018 ◽  
Vol 314 (6) ◽  
pp. H1298-H1308 ◽  
Author(s):  
Mary D. Frame ◽  
Anthony M. Dewar ◽  
Rhodora C. Calizo ◽  
Androniqi Qifti ◽  
Suzanne F. Scarlata
Keyword(s):  
Adrenergic Receptor ◽  
Low Dose ◽  
Nitrosative Stress ◽  
Fluorescent Protein ◽  
Receptor Internalization ◽  
Cheek Pouch ◽  
Red Fluorescent Protein ◽  
Hamster Cheek Pouch ◽  
Β2 Adrenergic Receptor ◽  
Inflammatory Insult

This study investigated the effect of sodium nitroprusside (SNP) preexposure on vasodilation via the β-adrenergic receptor (BAR) system. SNP was used as a nitrosative/oxidative proinflammatory insult. Small arterioles were visualized by intravital microscopy in the hamster cheek pouch tissue (isoflurane, n = 45). Control dilation to isoproterenol (EC50: 10−7 mol/l) became biphasic as a function of concentration after 2 min of exposure to SNP (10−4 M), with increased potency at picomolar dilation uncovered and decreased efficacy at the micromolar dilation. Control dilation to curcumin was likewise altered after SNP, but only the increased potency at a low dose was uncovered, whereas micromolar dilation was eliminated. The picomolar dilations were blocked by the potent BAR-2 inverse agonist carazolol (10−9 mol/l). Dynamin inhibition with dynasore mimicked this effect, suggesting that SNP preexposure prevented BAR agonist internalization. Using HeLa cells transfected with BAR-2 tagged with monomeric red fluorescent protein, exposure to 10−8−10−6 mol/l curcumin resulted in internalization and colocalization of BAR-2 and curcumin (FRET) that was prevented by oxidative stress (10−3 mol/l CoCl2), supporting that stress prevented internalization of the BAR agonist with the micromolar agonist. This study presents novel data supporting that distinct pools of BARs are differentially available after inflammatory insult. NEW & NOTEWORTHY Preexposure to an oxidative/nitrosative proinflammatory insult provides a “protective preconditioning” against future oxidative damage. We examined immediate vasoactive and molecular consequences of a brief preexposure via β-adrenergic receptor signaling in small arterioles. Blocked receptor internalization with elevated reactive oxygen levels coincides with a significant and unexpected vasodilation to β-adrenergic agonists at picomolar doses.

scholarly journals Red Fluorescent Protein (DsRed) as a Reporter inSaccharomyces cerevisiae

2001 ◽  
Vol 183 (12) ◽  
pp. 3791-3794 ◽  
Author(s):  
Fernando Rodrigues ◽  
Martijn van Hemert ◽  
H. Yde Steensma ◽  
Manuela Côrte-Real ◽  
Cecı́la Leão
Keyword(s):  
Nuclear Localization ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Green Fluorescent Proteins ◽  
Red Fluorescent Protein ◽  
Amino Terminal ◽  
Carboxyl Terminal ◽  
Dsred Protein ◽  
Green Fluorescent

ABSTRACT We describe the utilization of a red fluorescent protein (DsRed) as an in vivo marker for Saccharomyces cerevisiae. Clones expressing red and/or green fluorescent proteins with both cytoplasmic and nuclear localization were obtained. A series of vectors are now available which can be used to create amino-terminal (N-terminal) and carboxyl-terminal (C-terminal) fusions with the DsRed protein.

scholarly journals A red fluorescent protein with improved monomericity enables ratiometric voltage imaging with ASAP3

2020 ◽  
Author(s):  
Benjamin B. Kim ◽  
Haodi Wu ◽  
Yukun A. Hao ◽  
Michael Pan ◽  
Mariya Chavarha ◽  
...  
Keyword(s):  
High Performance ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
High Gain ◽  
Motion Artifacts ◽  
Red Fluorescent Protein ◽  
Red Fluorescent Proteins ◽  
Transmembrane Voltage ◽  
Site Mutagenesis ◽  
Green Fluorescent

AbstractA ratiometric genetically encoded voltage indicator (GEVI) would be desirable for tracking transmembrane voltage changes in cells that are undergoing motion. To create a high-performance ratiometric GEVI, we explored the possibility of adding a voltage-independent red fluorophore to ASAP3, a high-gain green fluorescent GEVI. We performed combinatorial multi-site mutagenesis on the cyan-excitable red fluorescent protein mCyRFP1 to enhance brightness and monomericity, creating mCyRFP3. Among red fluorescent proteins tested, mCyRFP3 proved to be the least perturbing when fused to ASAP3. We demonstrate that the red fluorescence of ASAP3-mCyRFP3 (ASAP3-R3) provides an effective reference channel to remove motion artifacts from voltage-induced changes in green fluorescence. Finally we use ASAP3-R3 to visualize membrane voltage changes throughout the cell cycle of motile cells.

20 GENERATION AND CHARACTERIZATION OF TRANSGENIC-CLONED PIGS EXPRESSING THE FAR-RED FLUORESCENT PROTEIN MONOMERIC PLUM

2014 ◽  
Vol 26 (1) ◽  
pp. 124
Author(s):  
M. Kobayashi ◽  
M. Watanabe ◽  
H. Matsunari ◽  
K. Nakano ◽  
T. Kanai ◽  
...  
Keyword(s):  
Mixed Culture ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Red Fluorescent Protein ◽  
Cell Stage ◽  
Fetal Fibroblasts ◽  
Morula Stage ◽  
Clear Identification ◽  
Green Fluorescent

Transgenic (Tg) pigs expressing a fluorescent protein are extremely useful for research into transplantation and regenerative medicine. This study aimed to create Tg pigs expressing monomeric Plum (mPlum), a far-red fluorescent protein with a longer wavelength than enhanced green fluorescent protein (EGFP) and humanized Kusabira Orange (huKO), the two fluorescent proteins that have been used previously for Tg pig production. A linearized CAG-mPlum transgene construct was transferred into porcine fetal fibroblasts (PFF) by electroporation. mPlum fluorescence-positive cells were collected using a cell sorter and used as nuclear donors (mPlum-PFF) for somatic cell nuclear transfer (SCNT). In vitro-matured oocytes were obtained from porcine cumulus–oocyte complexes cultured in NCSU23-based medium and were used to obtain recipient oocytes for SCNT after enucleation. Then, SCNT was performed as reported previously (Matsunari et al., 2008). The reconstructed embryos were cultured for 7 days in porcine zygote medium-5 (PZM-5). mPlum fluorescence expression was screened during the early development of the embryos. After 5 or 6 days of culture, the SCNT embryos were surgically transferred to the uterus of a recipient gilt. We first obtained fetuses on Day 36 or 37 of gestation by Caesarean section and the PFF were retrieved from their skin. Fluorescence expression was analysed using fluorescence microscope, and the number of transgene copies in each fetus was determined by Southern blot analysis. We also analysed whether unique spectral properties of mPlum are suitable for multicolor imaging using confocal microscope and flow cytometer. The identification of mPlum-expressing PFF under the mixed culture of PFF expressing EGFP and huKO was examined. The 2 cell lines of PFF expressing EGFP and huKO were previously generated in our laboratory. Rates of normal cleavage and blastocyst formation occurred in the SCNT embryos generated with mPlum-PFF (mPlum embryos) were equivalent to those of SCNT embryos derived from nontransgenic PFF (34/42, 81.0%; 33/42, 78.6% v. 37/40, 92.5%; 30/40, 75.0%). Total cell numbers in mPlum and control blastocysts did not differ significantly (88.3 ± 6.0 v. 99.9 ± 8.8). Fluorescence expression in the mPlum embryos began at the 8-cell stage and became brighter from the morula stage. The gilt into which 103 mPlum embryos were transferred produced 3 fetuses. These fetuses expressed mPlum fluorescence systemically and had 1 to 5 copies of the transgene. Multicolor fluorescence imaging and flow cytometric analyses of a mixed culture of mPlum PFF and PFF expressing EGFP and huKO showed that clear identification and isolation of cells displaying each of the 3 fluorescence signals was possible. These observations demonstrate that the transfer of CAG-mPlum did not interfere with the development of porcine SCNT embryos and resulted in the successful generation of Tg cloned pigs that systemically expressed mPlum. This work was supported by JSPS KAKENHI Grant Number 25293279.

Internal Trafficking and Surface Mobility of a Functionally Intact β2-Adrenergic Receptor-Green Fluorescent Protein Conjugate

1997 ◽  
Vol 51 (2) ◽  
pp. 177-184 ◽  
Author(s):  
Larry S. Barak ◽  
Stephen S. G. Ferguson ◽  
Jie Zhang ◽  
Christopher Martenson ◽  
Tobias Meyer ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Adrenergic Receptor ◽  
Fluorescent Protein ◽  
Surface Mobility ◽  
Protein Conjugate ◽  
Β2 Adrenergic Receptor ◽  
Green Fluorescent

Fluorescent proteins for in vivo imaging, where's the biliverdin?

2020 ◽  
Vol 48 (6) ◽  
pp. 2657-2667
Author(s):  
Felipe Montecinos-Franjola ◽  
John Y. Lin ◽  
Erik A. Rodriguez
Keyword(s):  
Hydrogen Peroxide ◽  
In Vivo Imaging ◽  
Near Infrared ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Fluorescent Imaging ◽  
Infrared Light ◽  
Red Fluorescent Protein ◽  
Color Palette

Noninvasive fluorescent imaging requires far-red and near-infrared fluorescent proteins for deeper imaging. Near-infrared light penetrates biological tissue with blood vessels due to low absorbance, scattering, and reflection of light and has a greater signal-to-noise due to less autofluorescence. Far-red and near-infrared fluorescent proteins absorb light >600 nm to expand the color palette for imaging multiple biosensors and noninvasive in vivo imaging. The ideal fluorescent proteins are bright, photobleach minimally, express well in the desired cells, do not oligomerize, and generate or incorporate exogenous fluorophores efficiently. Coral-derived red fluorescent proteins require oxygen for fluorophore formation and release two hydrogen peroxide molecules. New fluorescent proteins based on phytochrome and phycobiliproteins use biliverdin IXα as fluorophores, do not require oxygen for maturation to image anaerobic organisms and tumor core, and do not generate hydrogen peroxide. The small Ultra-Red Fluorescent Protein (smURFP) was evolved from a cyanobacterial phycobiliprotein to covalently attach biliverdin as an exogenous fluorophore. The small Ultra-Red Fluorescent Protein is biophysically as bright as the enhanced green fluorescent protein, is exceptionally photostable, used for biosensor development, and visible in living mice. Novel applications of smURFP include in vitro protein diagnostics with attomolar (10−18 M) sensitivity, encapsulation in viral particles, and fluorescent protein nanoparticles. However, the availability of biliverdin limits the fluorescence of biliverdin-attaching fluorescent proteins; hence, extra biliverdin is needed to enhance brightness. New methods for improved biliverdin bioavailability are necessary to develop improved bright far-red and near-infrared fluorescent proteins for noninvasive imaging in vivo.

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