Development of Fluorogenic Probes for Quick No-Wash Live-Cell Imaging of Intracellular Proteins

Hori, Yoshiaki; Norinobu, Tomoya; Sato, Motoki; Arita, Kyohei; Shirakawa, Masahiro; Kikuchi, Kazuya

doi:10.1021/ja405745v

Cited by 116 publications

(124 citation statements)

References 44 publications

Supporting

Mentioning

120

Contrasting

Order By: Relevance

“…Moreover, the binding pocket of PYP accommodates HC in its phenolate deprotonated form (25), providing a platform for designing variants able to stabilize deprotonated HBR and thus obtain absorption red shift upon binding. Finally, wild-type PYP has a proven ability as a recombinant protein tag (16,28) and is a small protein (14 kDa) compared with GFP-like fluorescent proteins (26−30 kDa).…”

Section: Resultsmentioning

confidence: 99%

Small fluorescence-activating and absorption-shifting tag for tunable protein imaging in vivo

Plamont

Billon-Denis

Maurin

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

223

353

View full text Add to dashboard Cite

This paper presents Yellow Fluorescence-Activating and absorption-Shifting Tag (Y-FAST), a small monomeric protein tag, half as large as the green fluorescent protein, enabling fluorescent labeling of proteins in a reversible and specific manner through the reversible binding and activation of a cell-permeant and nontoxic fluorogenic ligand (a so-called fluorogen). A unique fluorogen activation mechanism based on two spectroscopic changes, increase of fluorescence quantum yield and absorption red shift, provides high labeling selectivity. Y-FAST was engineered from the 14-kDa photoactive yellow protein by directed evolution using yeast display and fluorescence-activated cell sorting. Y-FAST is as bright as common fluorescent proteins, exhibits good photostability, and allows the efficient labeling of proteins in various organelles and hosts. Upon fluorogen binding, fluorescence appears instantaneously, allowing monitoring of rapid processes in near real time. Y-FAST distinguishes itself from other tagging systems because the fluorogen binding is highly dynamic and fully reversible, which enables rapid labeling and unlabeling of proteins by addition and withdrawal of the fluorogen, opening new exciting prospects for the development of multiplexing imaging protocols based on sequential labeling.

show abstract

Section: Resultsmentioning

confidence: 99%

Small fluorescence-activating and absorption-shifting tag for tunable protein imaging in vivo

Plamont

Billon-Denis

Maurin

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

223

353

View full text Add to dashboard Cite

show abstract

“…12 A protein-labeling technique has been recently developed using a PYP tag, in which the native chromophore of p-coumaric acid ( pCA) is substituted by the thioester derivative of cinnamic acid or coumarin through transthioesterification. [13][14][15][16] This alters the electronic structure in the excited state, protein cavity environment, and/or network of the intermolecular hydrogen bonding, and results in a novel fluorogenic probe for no-wash live-cell imaging of proteins fused to the PYP-tag. 14,15 Consequently, the fluorescence quantum yield increases significantly to 0.38-0.47, allowing a rapid detection of proteins in living cells with a high signal-to-noise ratio.…”

Section: Introductionmentioning

confidence: 99%

“…[13][14][15][16] This alters the electronic structure in the excited state, protein cavity environment, and/or network of the intermolecular hydrogen bonding, and results in a novel fluorogenic probe for no-wash live-cell imaging of proteins fused to the PYP-tag. 14,15 Consequently, the fluorescence quantum yield increases significantly to 0.38-0.47, allowing a rapid detection of proteins in living cells with a high signal-to-noise ratio. 14,15 Such a finding indicates that the photocycle mechanism for the isomerization and proton transfer reactions in the wild type (wt) PYP has been considerately changed due to the structural modification.…”

Section: Introductionmentioning

confidence: 99%

“…14,15 Consequently, the fluorescence quantum yield increases significantly to 0.38-0.47, allowing a rapid detection of proteins in living cells with a high signal-to-noise ratio. 14,15 Such a finding indicates that the photocycle mechanism for the isomerization and proton transfer reactions in the wild type (wt) PYP has been considerately changed due to the structural modification.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

A comprehensive study of isomerization and protonation reactions in the photocycle of the photoactive yellow protein

Wei

Wang

Chen

et al. 2014

Phys. Chem. Chem. Phys.

View full text Add to dashboard Cite

The light-activated photoactive yellow protein (PYP) chromophore uses a series of reactions to trigger photo-motility and biological responses, and generate a wide range of structural signals. To provide a comprehensive mechanism of the overall process at the atomic level, we apply a CASPT2//CASSCF/AMBER QM/MM protocol to investigate the relaxation pathways for a variety of possible isomerization and proton transfer reactions upon photoexcitation of the wild-type PYP. The nonadiabatic relay through an S1/S0 conical intersection [CI(S1/S0)] is found to play a decisive major role in bifurcating the excited state relaxation into a complete and short photocycle. Two major and one minor deactivation channels were found starting from the CI(S1/S0)-like intermediate IT, producing the cis isomers pR1, ICP, and ICT through "hula twist", "bicycle pedal" and one-bond flip isomerization reactions. The overall photocycle can be achieved by competitive parallel/sequential reactions, in which the ground state recovery is controlled by a series of slow volume-conserving bicycle pedal/hula twist and one-bond flip isomerization reactions, as well as fast protonation-deprotonation processes and the hydrophobic-hydrophilic state transformation.

show abstract

“…15 Recently, polarity-sensitive indicator of viability and apoptosis (pSIVA)-IANBD, an annexin-based, polaritysensitive dye for PS, has become available. 22 This indicator has no fluorescence in solution; however, it fluoresces strongly upon binding to PS on the cell surface, because of the change of its microenvironment from aqueous solution to lipid cell membrane, and this binding is reversible.…”

mentioning

confidence: 99%

Live-cell imaging to detect phosphatidylserine externalization in brain endothelial cells exposed to ionizing radiation: implications for the treatment of brain arteriovenous malformations

Zhao

Johnson

Chen

et al. 2016

JNS

View full text Add to dashboard Cite

Brain arteriovenous malformations (AVMs) are congenital abnormalities that consist of direct connections between arteries and veins, allowing highpressure arterial blood to flow into fragile cerebral veins, resulting in a high risk of hemorrhagic stroke. 16,37 The aim of AVM treatment is to prevent hemorrhage. 29 Treatment is effective only with complete AVM removal or obliteration. 14 Current treatments-namely surgery, endovascular occlusion, or stereotactic radiosurgery-are all associated with significant limitations.1,42 The surgical risk is generally unacceptable for lesions that are large, located in critical brain structures, or involve deep perforating arterabbreviatioNs AVM = arteriovenous malformation; FWD = forward; PI = propidium iodide; PS = phosphatidylserine; pSIVA = polarity-sensitive indicator of viability and apoptosis; SRS = stereotactic radiosurgery. obJective Stereotactic radiosurgery (SRS) is an established intervention for brain arteriovenous malformations (AVMs). The processes of AVM vessel occlusion after SRS are poorly understood. To improve SRS efficacy, it is important to understand the cellular response of blood vessels to radiation. The molecular changes on the surface of AVM endothelial cells after irradiation may also be used for vascular targeting. This study investigates radiation-induced externalization of phosphatidylserine (PS) on endothelial cells using live-cell imaging. methods An immortalized cell line generated from mouse brain endothelium, bEnd.3 cells, was cultured and irradiated at different radiation doses using a linear accelerator. PS externalization in the cells was subsequently visualized using polarity-sensitive indicator of viability and apoptosis (pSIVA)-IANBD, a polarity-sensitive probe. Live-cell imaging was used to monitor PS externalization in real time. The effects of radiation on the cell cycle of bEnd.3 cells were also examined by flow cytometry. results Ionizing radiation effects are dose dependent. Reduction in the cell proliferation rate was observed after exposure to 5 Gy radiation, whereas higher radiation doses (15 Gy and 25 Gy) totally inhibited proliferation. In comparison with cells treated with sham radiation, the irradiated cells showed distinct pseudopodial elongation with little or no spreading of the cell body. The percentages of pSIVA-positive cells were significantly higher (p = 0.04) 24 hours after treatment in the cultures that received 25- and 15-Gy doses of radiation. This effect was sustained until the end of the experiment (3 days). Radiation at 5 Gy did not induce significant PS externalization compared with the sham-radiation controls at any time points (p > 0.15). Flow cytometric analysis data indicate that irradiation induced growth arrest of bEnd.3 cells, with cells accumulating in the G2 phase of the cell cycle. coNclusioNs Ionizing radiation causes remarkable cellular changes in endothelial cells. Significant PS externalization is induced by radiation at doses of 15 Gy or higher, concomitant with a block in the cell cycle. Ra...

show abstract

Development of Fluorogenic Probes for Quick No-Wash Live-Cell Imaging of Intracellular Proteins

Cited by 116 publications

References 44 publications

Small fluorescence-activating and absorption-shifting tag for tunable protein imaging in vivo

Small fluorescence-activating and absorption-shifting tag for tunable protein imaging in vivo

A comprehensive study of isomerization and protonation reactions in the photocycle of the photoactive yellow protein

Live-cell imaging to detect phosphatidylserine externalization in brain endothelial cells exposed to ionizing radiation: implications for the treatment of brain arteriovenous malformations

Contact Info

Product

Resources

About