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

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

AJP Cell Physiology ◽

10.1152/ajpcell.00291.2006 ◽

2007 ◽

Vol 292 (2) ◽

pp. C814-C823 ◽

Cited By ~ 15

Author(s):

Kerry J. Rhoden ◽

Stefano Cianchetta ◽

Valeria Stivani ◽

Carla Portulano ◽

Luis J. V. Galietta ◽

...

Keyword(s):

Live Cell Imaging ◽

Sodium Iodide ◽

Cell Imaging ◽

Fluorescent Protein ◽

Yellow Fluorescent Protein ◽

Nuclear Imaging ◽

Sodium Iodide Symporter ◽

Protein Variant ◽

Thyroid Cells

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 Förster Resonance Energy Transfer-Based Sensing Strategies

ACS Sensors ◽

10.1021/acssensors.1c00874 ◽

2021 ◽

Author(s):

Yasmina Bousmah ◽

Hana Valenta ◽

Giulia Bertolin ◽

Utkarsh Singh ◽

Valérie Nicolas ◽

...

Keyword(s):

Energy Transfer ◽

Live Cell Imaging ◽

Cell Imaging ◽

Fluorescent Protein ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Live Cell ◽

Forster Resonance Energy Transfer ◽

Förster Resonance Energy Transfer ◽

Förster Resonance

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Single-molecule live-cell imaging of bacterial DNA repair and damage tolerance

Biochemical Society Transactions ◽

10.1042/bst20170055 ◽

2017 ◽

Vol 46 (1) ◽

pp. 23-35 ◽

Cited By ~ 7

Author(s):

Harshad Ghodke ◽

Han Ho ◽

Antoine M. van Oijen

Keyword(s):

Dna Repair ◽

Single Molecule ◽

Live Cell Imaging ◽

Spatial Organization ◽

Cell Imaging ◽

Live Cell ◽

Live Cells ◽

Temporal Regulation ◽

Repair Processes ◽

Wide Range

Genomic DNA is constantly under threat from intracellular and environmental factors that damage its chemical structure. Uncorrected DNA damage may impede cellular propagation or even result in cell death, making it critical to restore genomic integrity. Decades of research have revealed a wide range of mechanisms through which repair factors recognize damage and co-ordinate repair processes. In recent years, single-molecule live-cell imaging methods have further enriched our understanding of how repair factors operate in the crowded intracellular environment. The ability to follow individual biochemical events, as they occur in live cells, makes single-molecule techniques tremendously powerful to uncover the spatial organization and temporal regulation of repair factors during DNA–repair reactions. In this review, we will cover practical aspects of single-molecule live-cell imaging and highlight recent advances accomplished by the application of these experimental approaches to the study of DNA–repair processes in prokaryotes.

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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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Live-Cell Imaging of Rhabdovirus-Induced Morphological Changes in Plant Nuclear Membranes

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-18-0703 ◽

2005 ◽

Vol 18 (7) ◽

pp. 703-709 ◽

Cited By ~ 32

Author(s):

Michael Goodin ◽

Sharon Yelton ◽

Debasish Ghosh ◽

Stephanie Mathews ◽

Judith Lesnaw

Keyword(s):

Live Cell Imaging ◽

Cell Imaging ◽

Fluorescent Protein ◽

Morphological Changes ◽

Live Cell ◽

Live Cells ◽

Nuclear Membranes ◽

Yellow Dwarf Virus ◽

Study Sites ◽

Green Fluorescent

Potato yellow dwarf virus (PYDV) and Sonchus yellow net virus (SYNV) belong to the genus Nucleorhabdovirus. These viruses replicate in nuclei of infected cells and mature virions accumulate in the perinuclear space after budding through the inner nuclear membrane. Infection of transgenic Nicotiana benthamiana 16c plants (which constitutively express green fluorescent protein (GFP) targeted to endomembranes) with PYDV or SYNV resulted in virusspecific patterns of accumulation of both GFP and membranes within nuclei. Using immunolocalization and a lipophilic fluorescent dye, we show that the sites of the relocalized membranes were coincident with foci of accumulation of the SYNV nucleocapsid protein. In contrast to the effects of PYDV and SYNV, inoculation of 16c plants with plusstrand RNA viruses did not result in accumulation of intranuclear GFP. Instead, such infections resulted in accumulation of GFP around nuclei, in a manner consistent with proliferation of the endoplasmic reticulum. We propose that the relocalization of GFP in 16c plants can be used to study sites of rhabdovirus accumulation in live cells. This study is the first to use live-cell imaging to characterize the effects of rhabdoviruses on plant nuclear membranes.

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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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