scholarly journals Deuteration of terminal alkynes realizes simultaneous live cell Raman imaging of similar alkyne-tagged biomolecules

2021 ◽  
Author(s):  
Syusuke Egoshi ◽  
Kosuke Dodo ◽  
Kenji Ohgane ◽  
Mikiko Sodeoka
Keyword(s):  
Small Molecules ◽  
Raman Imaging ◽  
Live Cell ◽  
Live Cells ◽  
Terminal Alkynes

Two-color Raman imaging of D-alkynes and H-alkynes makes it possible to distinguish and observe similar small molecules in live cells.

Alkyne-tag Raman imaging of bio-active small molecules in live cells

2015 ◽  
Author(s):  
Jun Ando ◽  
Almar F. Palonpon ◽  
Hiroyuki Yamakoshi ◽  
Kosuke Dodo ◽  
Satoshi Kawata ◽  
...  
Keyword(s):  
Small Molecules ◽  
Raman Imaging ◽  
Live Cells

Alkyne-Tag Raman Imaging for Visualization of Mobile Small Molecules in Live Cells

2012 ◽  
Vol 134 (51) ◽  
pp. 20681-20689 ◽  
Author(s):  
Hiroyuki Yamakoshi ◽  
Kosuke Dodo ◽  
Almar Palonpon ◽  
Jun Ando ◽  
Katsumasa Fujita ◽  
...  
Keyword(s):  
Small Molecules ◽  
Raman Imaging ◽  
Live Cells

Watching bioactive small molecules in live cells by alkyne-tag Raman imaging

2013 ◽  
Author(s):  
Almar Palonpon ◽  
Hiroyuki Yamakoshi ◽  
Kosuke Dodo ◽  
Jun Ando ◽  
Satoshi Kawata ◽  
...  
Keyword(s):  
Small Molecules ◽  
Raman Imaging ◽  
Live Cells

α-Methylene-β-Lactone Probe for Measuring Live-Cell Reactions of Small Molecules

2020 ◽  
Author(s):  
Lei Wang ◽  
Louis Riel ◽  
Bekim Bajrami ◽  
Bin Deng ◽  
Amy Howell ◽  
...  
Keyword(s):  
Mass Spectra ◽  
Live Cell ◽  
Live Cells ◽  
The Novel ◽  
Proteomics Analysis ◽  
Chemical Proteomics ◽  
Tandem Mass Spectra ◽  
Modified Peptides ◽  
Covalent Probes ◽  
Ht 29

The novel use of the α-methylene-β-lactone (MeLac) moiety as a warhead of multiple electrophilic sites is reported. In this study, we demonstrate that a MeLac-alkyne is a competent covalent probe and reacts with diverse proteins in live cells. Proteomics analysis of affinity-enriched samples identifies probe-reacted proteins, resolves their modified peptides/residues, and thus characterizes probe-protein reactions. Unique methods are developed to evaluate confidence in the identification of the reacted proteins and modified peptides. Tandem mass spectra of the peptides reveal that MeLac reacts with nucleophilic cysteine, serine, lysine, threonine, and tyrosine residues, through either Michael addition or acyl addition. A peptide-centric proteomics platform, using MeLac-alkyne as the measurement probe, successfully analyzes the Orlistat selectivity in live HT-29 cells. MeLac is a versatile warhead demonstrating enormous potential to expedite the development of covalent probes and inhibitors in interrogating protein (re)activity. MeLac-empowered platforms in chemical proteomics are widely adaptable for measuring the live-cell action of reactive molecules.

scholarly journals Fluorescent Probes for Live Cell Thiol Detection

Molecules ◽  
2021 ◽  
Vol 26 (12) ◽  
pp. 3575
Author(s):  
Shenggang Wang ◽  
Yue Huang ◽  
Xiangming Guan
Keyword(s):  
Fluorescent Probes ◽  
Biological System ◽  
High Sensitivity ◽  
Intracellular Distribution ◽  
Live Cell ◽  
Live Cells ◽  
High Demand ◽  
Non Invasive

Thiols play vital and irreplaceable roles in the biological system. Abnormality of thiol levels has been linked with various diseases and biological disorders. Thiols are known to distribute unevenly and change dynamically in the biological system. Methods that can determine thiols’ concentration and distribution in live cells are in high demand. In the last two decades, fluorescent probes have emerged as a powerful tool for achieving that goal for the simplicity, high sensitivity, and capability of visualizing the analytes in live cells in a non-invasive way. They also enable the determination of intracellular distribution and dynamitic movement of thiols in the intact native environments. This review focuses on some of the major strategies/mechanisms being used for detecting GSH, Cys/Hcy, and other thiols in live cells via fluorescent probes, and how they are applied at the cellular and subcellular levels. The sensing mechanisms (for GSH and Cys/Hcy) and bio-applications of the probes are illustrated followed by a summary of probes for selectively detecting cellular and subcellular thiols.

Novel anthracene- and pyridine-containing Schiff base probe for selective “off–on” fluorescent determination of Cu2+ ions towards live cell application

2016 ◽  
Vol 40 (7) ◽  
pp. 6101-6108 ◽  
Author(s):  
Turibius Simon ◽  
Muthaiah Shellaiah ◽  
Venkatesan Srinivasadesikan ◽  
Ching-Chang Lin ◽  
Fu-Hsiang Ko ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Schiff Base ◽  
Photoinduced Electron Transfer ◽  
Transfer Mechanism ◽  
Live Cell ◽  
Live Cells ◽  
Electron Transfer Mechanism

A simple anthracene-based AP probe was synthesized to detect Cu2+ ions, via the photoinduced electron transfer mechanism, in live cells.

scholarly journals Characterization of a Replicating Mammalian Orthoreovirus with Tetracysteine-Tagged μNS for Live-Cell Visualization of Viral Factories

2017 ◽  
Vol 91 (22) ◽  
Author(s):  
Luke D. Bussiere ◽  
Promisree Choudhury ◽  
Bryan Bellaire ◽  
Cathy L. Miller
Keyword(s):  
Nonstructural Protein ◽  
Critical Role ◽  
Viral Proteins ◽  
Live Cell ◽  
Host Cells ◽  
Live Cells ◽  
Virus Protein ◽  
Mammalian Orthoreovirus ◽  
Virus Proteins

ABSTRACT Within infected host cells, mammalian orthoreovirus (MRV) forms viral factories (VFs), which are sites of viral transcription, translation, assembly, and replication. The MRV nonstructural protein μNS comprises the structural matrix of VFs and is involved in recruiting other viral proteins to VF structures. Previous attempts have been made to visualize VF dynamics in live cells, but due to current limitations in recovery of replicating reoviruses carrying large fluorescent protein tags, researchers have been unable to directly assess VF dynamics from virus-produced μNS. We set out to develop a method to overcome this obstacle by utilizing the 6-amino-acid (CCPGCC) tetracysteine (TC) tag and FlAsH-EDT2 reagent. The TC tag was introduced into eight sites throughout μNS, and the capacity of the TC-μNS fusion proteins to form virus factory-like (VFL) structures and colocalize with virus proteins was characterized. Insertion of the TC tag interfered with recombinant virus rescue in six of the eight mutants, likely as a result of loss of VF formation or important virus protein interactions. However, two recombinant (r)TC-μNS viruses were rescued and VF formation, colocalization with associating virus proteins, and characterization of virus replication were subsequently examined. Furthermore, the rTC-μNS viruses were utilized to infect cells and examine VF dynamics using live-cell microscopy. These experiments demonstrate active VF movement with fusion events as well as transient interactions between individual VFs and demonstrate the importance of microtubule stability for VF fusion during MRV infection. This work provides important groundwork for future in-depth studies of VF dynamics and host cell interactions. IMPORTANCE MRV has historically been used as a model to study the double-stranded RNA (dsRNA) Reoviridae family, the members of which infect and cause disease in humans, animals, and plants. During infection, MRV forms VFs that play a critical role in virus infection but remain to be fully characterized. To study VFs, researchers have focused on visualizing the nonstructural protein μNS, which forms the VF matrix. This work provides the first evidence of recovery of replicating reoviruses in which VFs can be labeled in live cells via introduction of a TC tag into the μNS open reading frame. Characterization of each recombinant reovirus sheds light on μNS interactions with viral proteins. Moreover, utilizing the TC-labeling FlAsH-EDT2 biarsenical reagent to visualize VFs, evidence is provided of dynamic VF movement and interactions at least partially dependent on intact microtubules.

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

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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