Cell-based imaging of sodium iodide symporter activity with the yellow fluorescent protein variant YFP-H148Q/I152L

The sodium iodide symporter (NIS) mediates iodide (I−) transport in the thyroid gland and other tissues and is of increasing importance as a therapeutic target and nuclear imaging reporter. NIS activity in vitro is currently measured with radiotracers and electrophysiological techniques. We report on the development of a novel live cell imaging assay of NIS activity using the I−-sensitive and genetically encodable yellow fluorescent protein (YFP) variant YFP-H148Q/I152L. In FRTL-5 thyrocytes stably expressing YFP-H148Q/I152L, I− induced a rapid and reversible decrease in cellular fluorescence characterized by 1) high affinity for extracellular I− (35 μM), 2) inhibition by the NIS inhibitor perchlorate, 3) extracellular Na+ dependence, and 4) TSH dependence, suggesting that fluorescence changes are due to I− influx via NIS. Individual cells within a population of FRTL-5 cells exhibited a 3.5-fold variation in the rate of NIS-mediated I− influx, illustrating the utility of YFP-H148Q/I152L to detect cell-to-cell difference in NIS activity. I− also caused a perchlorate-sensitive decrease in YFP-H148Q/I152L fluorescence in COS-7 cells expressing NIS but not in cells lacking NIS. These results demonstrate that YFP-H148Q/I152L is a sensitive biosensor of NIS-mediated I− uptake in thyroid cells and in nonthyroidal cells following gene transfer and suggest that fluorescence detection of cellular I− may be a useful tool by which to study the pathophysiology and pharmacology of NIS.

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tdLanYFP, a yellow, bright, photostable and pH insensitive fluorescent protein for live cell imaging and FRET-based sensing strategies

10.1101/2021.04.27.441613 ◽

2021 ◽

Author(s):

Y. Bousmah ◽

H. Valenta ◽

G. Bertolin ◽

U. Singh ◽

V. Nicolas ◽

...

Keyword(s):

Single Molecule ◽

Live Cell Imaging ◽

Cell Imaging ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

Yellow Fluorescent Protein ◽

Resonance Energy ◽

Live Cell ◽

Live Cells

AbstractYellow fluorescent proteins (YFP) are widely used as optical reporters in Förster Resonance Energy Transfer (FRET) based biosensors. Although great improvements have been done, the sensitivity of the biosensors is still limited by the low photostability and the poor fluorescence performances of YFPs at acidic pHs. In fact, today, there is no yellow variant derived from the EYFP with a pK1/2 below ∼5.5. Here, we characterize a new yellow fluorescent protein, tdLanYFP, derived from the tetrameric protein from the cephalochordate B. lanceolatum, LanYFP. With a quantum yield of 0.92 and an extinction coefficient of 133 000 mol−1.L.cm−1, it is, to our knowledge, the brightest dimeric fluorescent protein available, and brighter than most of the monomeric YFPs. Contrasting with EYFP and its derivatives, tdLanYFP has a very high photostability in vitro and preserves this property in live cells. As a consequence, tdLanYFP allows the imaging of cellular structures with sub-diffraction resolution with STED nanoscopy. We also demonstrate that the combination of high brightness and strong photostability is compatible with the use of spectro-microscopies in single molecule regimes. Its very low pK1/2 of 3.9 makes tdLanYFP an excellent tag even at acidic pHs. Finally, we show that tdLanYFP can be a FRET partner either as donor or acceptor in different biosensing modalities. Altogether, these assets make tdLanYFPa very attractive yellow fluorescent protein for long-term or single-molecule live-cell imaging that is also suitable for FRET experiment including at acidic pH.

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Perchlorate transport and inhibition of the sodium iodide symporter measured with the yellow fluorescent protein variant YFP-H148Q/I152L

Toxicology and Applied Pharmacology ◽

10.1016/j.taap.2009.12.004 ◽

2010 ◽

Vol 243 (3) ◽

pp. 372-380 ◽

Cited By ~ 19

Author(s):

Stefano Cianchetta ◽

Julie di Bernardo ◽

Giovanni Romeo ◽

Kerry J. Rhoden

Keyword(s):

Sodium Iodide ◽

Fluorescent Protein ◽

Yellow Fluorescent Protein ◽

Sodium Iodide Symporter ◽

Protein Variant

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Forskolin, 8-Br-3′,5′-Cyclic Adenosine 5′-Monophosphate, and Catalytic Protein Kinase A Expression in the Nucleus Increase Radioiodide Uptake and Sodium/Iodide Symporter Protein Levels in RET/PTC1-Expressing Cells

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/jc.2004-1414 ◽

2004 ◽

Vol 89 (12) ◽

pp. 6168-6172 ◽

Cited By ~ 13

Author(s):

Anjli Venkateswaran ◽

Derek K. Marsee ◽

Steven H. Green ◽

Sissy M. Jhiang

Keyword(s):

Protein Kinase ◽

Protein Kinase A ◽

Sodium Iodide ◽

Sodium Iodide Symporter ◽

Thyroid Cells ◽

Protein Levels ◽

And Function ◽

Kinase A

Abstract RET/PTC1, a thyroid-specific oncogene, has been reported to down-regulate sodium/iodide symporter (NIS) expression and function in vitro and in vivo. Recently, RET/PTC1 has been shown to interfere with TSH signaling at multiple levels in thyroid cells. The objective of this study was to investigate whether RET/PTC1-mediated NIS reduction can be rescued by activating cAMP-protein kinase A (PKA) pathways. We showed that both forskolin and 8-Br-cAMP increase radioiodide uptake and NIS protein in RET/PTC1-expressing cells to the same extent as the parental PC Cl 3 cells. We found that RET/PTC1 decreases nuclear localization of catalytic PKA, and forskolin treatment was able to counteract this RET/PTC1 effect. Furthermore, transient expression of catalytic PKA in the nucleus increased radioiodide uptake and NIS protein in RET/PTC1-expressing cells. Taken together, these studies suggest that RET/PTC1 down-regulates NIS expression by interrupting TSH/cAMP signaling, and this RET/PTC1 effect can be reversed by activating cAMP-PKA pathways.

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Vectorial insertion of apical and basolateral membrane proteins in polarized epithelial cells revealed by quantitative 3D live cell imaging

The Journal of Cell Biology ◽

10.1083/jcb.200512012 ◽

2006 ◽

Vol 172 (7) ◽

pp. 1035-1044 ◽

Cited By ~ 43

Author(s):

Wei Hua ◽

David Sheff ◽

Derek Toomre ◽

Ira Mellman

Keyword(s):

Membrane Proteins ◽

Epithelial Cells ◽

Live Cell Imaging ◽

Cell Imaging ◽

Fluorescent Protein ◽

Yellow Fluorescent Protein ◽

Basolateral Membrane ◽

Live Cell ◽

Junctional Complex ◽

Apical Surface

Although epithelial cells are known to exhibit a polarized distribution of membrane components, the pathways responsible for delivering membrane proteins to their appropriate domains remain unclear. Using an optimized approach to three-dimensional live cell imaging, we have visualized the transport of newly synthesized apical and basolateral membrane proteins in fully polarized filter-grown Madin–Darby canine kidney cells. We performed a detailed quantitative kinetic analysis of trans-Golgi network (TGN) exit, passage through transport intermediates, and arrival at the plasma membrane using cyan/yellow fluorescent protein–tagged glycosylphosphatidylinositol-anchored protein and vesicular stomatitis virus glycoprotein as apical and basolateral reporters, respectively. For both pathways, exit from the TGN was rate limiting. Furthermore, apical and basolateral proteins were targeted directly to their respective membranes, resolving current confusion as to whether sorting occurs on the secretory pathway or only after endocytosis. However, a transcytotic protein did reach the apical surface after a prior appearance basolaterally. Finally, newly synthesized proteins appeared to be delivered to the entire lateral or apical surface, suggesting—contrary to expectations—that there is not a restricted site for vesicle docking or fusion adjacent to the junctional complex.

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Near-infrared imaging in fission yeast by genetically encoded biosynthesis of phycocyanobilin

Journal of Cell Science ◽

10.1242/jcs.259315 ◽

2021 ◽

Author(s):

Keiichiro Sakai ◽

Yohei Kondo ◽

Hiroyoshi Fujioka ◽

Mako Kamiya ◽

Kazuhiro Aoki ◽

...

Keyword(s):

Fission Yeast ◽

Live Cell Imaging ◽

Near Infrared ◽

Cell Imaging ◽

Infrared Imaging ◽

Fluorescent Protein ◽

Live Cell ◽

Yeast Cells ◽

Color Palette

Near-infrared fluorescent protein (iRFP) is a bright and stable fluorescent protein with near-infrared excitation and emission maxima. Unlike the other conventional fluorescent proteins, iRFP requires biliverdin (BV) as a chromophore. Here, we report that phycocyanobilin (PCB) functions as a brighter chromophore for iRFP than BV, and biosynthesis of PCB allows live-cell imaging with iRFP in the fission yeast Schizosaccharomyces pombe. We initially found that fission yeast cells did not produce BV, and therefore did not show any iRFP fluorescence. The brightness of iRFP-PCB was higher than that of iRFP-BV in vitro and in fission yeast. We introduced SynPCB, a PCB biosynthesis system, into fission yeast, resulting in the brightest iRFP fluorescence. To make iRFP readily available in fission yeast, we developed an endogenous gene tagging system with iRFP and all-in-one integration plasmids carrying the iRFP-fused marker proteins together with SynPCB. These tools not only enable the easy use of the multiplexed live-cell imaging in fission yeast with a broader color palette, but also open the door to new opportunities for near-infrared fluorescence imaging in a wider range of living organisms.

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Near-infrared imaging in fission yeast by genetically encoded biosynthesis of phycocyanobilin

10.1101/2021.05.19.444883 ◽

2021 ◽

Author(s):

Keiichiro Sakai ◽

Yohei Kondo ◽

Hiroyoshi Fujioka ◽

Mako Kamiya ◽

Kazuhiro Aoki ◽

...

Keyword(s):

Fission Yeast ◽

Live Cell Imaging ◽

Near Infrared ◽

Cell Imaging ◽

Infrared Imaging ◽

Fluorescent Protein ◽

Live Cell ◽

Yeast Cells ◽

Color Palette

Near-infrared fluorescent protein (iRFP) is the bright and stable fluorescent protein with excitation and emission maxima at 690 nm and 713 nm, respectively. Unlike the other conventional fluorescent proteins such as GFP, iRFP requires biliverdin (BV) as a chromophore because iRFP originates from phytochrome. Here, we report that phycocyanobilin (PCB) functions as a brighter chromophore for iRFP than BV, and biosynthesis of PCB allows live-cell imaging with iRFP in fission yeast Schizosaccharomyces pombe. We initially found that fission yeast cells did not produce BV, and therefore did not show any iRFP fluorescence. The brightness of iRFP attached to PCB was higher than that attached to BV in vitro and in fission yeast. We introduced SynPCB, a previously reported PCB biosynthesis system, into fission yeast, resulting in the brightest iRFP fluorescence. To make iRFP readily available in fission yeast, we developed an endogenous gene tagging system with iRFP and all-in-one integration plasmids, which contain genes required for the SynPCB system and the iRFP-fused marker proteins. These tools not only enable the easy use of iRFP in fission yeast and the multiplexed live-cell imaging in fission yeast with a broader color palette, but also open the doors to new opportunities for near-infrared fluorescence imaging in a wider range of living organisms.

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The CRISPR/Cas9 Minipig—A Transgenic Minipig to Produce Specific Mutations in Designated Tissues

Cancers ◽

10.3390/cancers13123024 ◽

2021 ◽

Vol 13 (12) ◽

pp. 3024

Author(s):

Martin Fogtmann Berthelsen ◽

Maria Riedel ◽

Huiqiang Cai ◽

Søren H. Skaarup ◽

Aage K. O. Alstrup ◽

...

Keyword(s):

Fluorescent Protein ◽

Yellow Fluorescent Protein ◽

Somatic Cells ◽

Genetic Alterations ◽

Lung Epithelial Cells ◽

Target Cells ◽

Multiple Gene ◽

Conditional Expression ◽

Gene Alterations

The generation of large transgenic animals is impeded by complex cloning, long maturation and gastrulation times. An introduction of multiple gene alterations increases the complexity. We have cloned a transgenic Cas9 minipig to introduce multiple mutations by CRISPR in somatic cells. Transgenic Cas9 pigs were generated by somatic cell nuclear transfer and were backcrossed to Göttingen Minipigs for two generations. Cas9 expression was controlled by FlpO-mediated recombination and was visualized by translation from red to yellow fluorescent protein. In vitro analyses in primary fibroblasts, keratinocytes and lung epithelial cells confirmed the genetic alterations executed by the viral delivery of single guide RNAs (sgRNA) to the target cells. Moreover, multiple gene alterations could be introduced simultaneously in a cell by viral delivery of sgRNAs. Cells with loss of TP53, PTEN and gain-of-function mutation in KRASG12D showed increased proliferation, confirming a transformation of the primary cells. An in vivo activation of Cas9 expression could be induced by viral delivery to the skin. Overall, we have generated a minipig with conditional expression of Cas9, where multiple gene alterations can be introduced to somatic cells by viral delivery of sgRNA. The development of a transgenic Cas9 minipig facilitates the creation of complex pre-clinical models for cancer research.

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Red-Shifted Aminated Derivatives of GFP Chromophore for Live-Cell Protein Labeling with Lipocalins

International Journal of Molecular Sciences ◽

10.3390/ijms19123778 ◽

2018 ◽

Vol 19 (12) ◽

pp. 3778 ◽

Cited By ~ 5

Author(s):

Nina Bozhanova ◽

Mikhail Baranov ◽

Nadezhda Baleeva ◽

Alexey Gavrikov ◽

Alexander Mishin

Keyword(s):

Green Fluorescent Protein ◽

Live Cell Imaging ◽

Cell Imaging ◽

Fluorescent Protein ◽

Live Cell ◽

Protein Labeling ◽

Cell Protein ◽

Gfp Chromophore ◽

Green Fluorescent ◽

Derivatives Of

Fluorogens are an attractive type of dye for imaging applications, eliminating time-consuming washout steps from staining protocols. With just a handful of reported fluorogen-protein pairs, mostly in the green region of spectra, there is a need for the expansion of their spectral range. Still, the origins of solvatochromic and fluorogenic properties of the chromophores suitable for live-cell imaging are poorly understood. Here we report on the synthesis and labeling applications of novel red-shifted fluorogenic cell-permeable green fluorescent protein (GFP) chromophore analogs.

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Identity of the renin cell is mediated by cAMP and chromatin remodeling: an in vitro model for studying cell recruitment and plasticity

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.01152.2007 ◽

2008 ◽

Vol 294 (2) ◽

pp. H699-H707 ◽

Cited By ~ 46

Author(s):

Ellen Steward Pentz ◽

Maria Luisa S. Sequeira Lopez ◽

Magali Cordaillat ◽

R. Ariel Gomez

Keyword(s):

Chromatin Remodeling ◽

In Vitro Model ◽

Molecular Mechanisms ◽

Fluorescent Protein ◽

Yellow Fluorescent Protein ◽

Histone H4 Acetylation ◽

Renin Gene ◽

Vitro Model

The renin-angiotensin system (RAS) regulates blood pressure and fluid-electrolyte homeostasis. A key step in the RAS cascade is the regulation of renin synthesis and release by the kidney. We and others have shown that a major mechanism to control renin availability is the regulation of the number of cells capable of making renin. The kidney possesses a pool of cells, mainly in its vasculature but also in the glomeruli, capable of switching from smooth muscle to endocrine renin-producing cells when homeostasis is threatened. The molecular mechanisms governing the ability of these cells to turn the renin phenotype on and off have been very difficult to study in vivo. We, therefore, developed an in vitro model in which cells of the renin lineage are labeled with cyan fluorescent protein and cells actively making renin mRNA are labeled with yellow fluorescent protein. The model allowed us to determine that it is possible to culture cells of the renin lineage for numerous passages and that the memory to express the renin gene is maintained in culture and can be reenacted by cAMP and chromatin remodeling (histone H4 acetylation) at the cAMP-responsive element in the renin gene.

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