Live Cell Imaging of Enzymatic Turnover of an Adenosine 5′-Tetraphosphate Analog

The hydrolysis of nucleotides is of paramount importance as an energy source for cellular processes. In addition, the transfer of phosphates from nucleotides onto proteins is important as a post-translational protein modification. Monitoring the enzymatic turnover of nucleotides therefore offers great potential as a tool to follow enzymatic activity. While a number of fluorescence sensors are known, so far, there are no methods available for the real-time monitoring of ATP hydrolysis inside live cells. We present the synthesis and application of a novel fluorogenic adenosine 5′-tetraphosphate (Ap4) analog suited for this task. Upon enzymatic hydrolysis, the molecule displays an increase in fluorescence intensity, which provides a readout of its turnover. We demonstrate how this can be used for monitoring cellular processes involving Ap4 hydrolysis. To this end, we visualized the enzymatic activity in live cells using confocal fluorescence microscopy of the Ap4 analog. Our results demonstrate that the Ap4 analog is hydrolyzed in lysosomes. We show that this approach is suited to visualize the lysosome distribution profiles within the live cell and discuss how it can be employed to gather information regarding autophagic flux.

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Live Cell Imaging of Single RNA Molecules with Fluorogenic Mango II Arrays

10.1101/840082 ◽

2019 ◽

Author(s):

Adam D. Cawte ◽

Peter J. Unrau ◽

David S. Rueda

Keyword(s):

Single Molecule ◽

Signal To Noise Ratio ◽

Super Resolution ◽

Live Cell ◽

Rna Aptamers ◽

Live Cells ◽

Structured Illumination ◽

Rna Molecules ◽

Cellular Processes ◽

Rna Imaging

AbstractRNA molecules play vital roles in many cellular processes. Visualising their dynamics in live cells at single-molecule resolution is essential to elucidate their role in RNA metabolism. RNA aptamers, such as Spinach and Mango, have recently emerged as a powerful background-free technology for live-cell RNA imaging due to their fluorogenic properties upon ligand binding. Here, we report a novel array of Mango II aptamers for RNA imaging in live and fixed cells with high contrast and single-molecule sensitivity. Direct comparison of Mango II and MS2-tdMCP-mCherry dual-labelled mRNAs show marked improvements in signal to noise ratio using the fluorogenic Mango aptamers. Using both coding (β-actin mRNA) and long non-coding (NEAT1) RNAs, we show that the Mango array does not affect cellular localisation. Additionally, we can track single mRNAs for extended time periods, likely due to fluorophore exchange. This property makes the arrays readily compatible with structured illumination super-resolution microscopy.

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

10.26434/chemrxiv.12078582.v2 ◽

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.

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Faculty Opinions recommendation of Monoglyceride lipase-like enzymatic activity is responsible for hydrolysis of 2-arachidonoylglycerol in rat cerebellar membranes.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1018005.207560 ◽

2004 ◽

Author(s):

Nephi Stella

Keyword(s):

Enzymatic Activity ◽

Monoglyceride Lipase ◽

Hydrolysis Of

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Functional Fluorescence Microscopy Imaging: Quantitative Scanning-Free Confocal Fluorescence Microscopy for the Characterization of Fast Dynamic Processes in Live Cells

Analytical Chemistry ◽

10.1021/acs.analchem.9b01813 ◽

2019 ◽

Vol 91 (17) ◽

pp. 11129-11137 ◽

Cited By ~ 5

Author(s):

Aleksandar J. Krmpot ◽

Stanko N. Nikolić ◽

Sho Oasa ◽

Dimitrios K. Papadopoulos ◽

Marco Vitali ◽

...

Keyword(s):

Fluorescence Microscopy ◽

Confocal Fluorescence Microscopy ◽

Live Cells ◽

Dynamic Processes ◽

Microscopy Imaging ◽

Confocal Fluorescence ◽

Fast Dynamic

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Hydrolysis of nucleoside triphosphates catalyzed by the recA protein of Escherichia coli. Steady state kinetic analysis of ATP hydrolysis.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)68922-2 ◽

1981 ◽

Vol 256 (16) ◽

pp. 8845-8849 ◽

Cited By ~ 8

Author(s):

G.M. Weinstock ◽

K. McEntee ◽

I.R. Lehman

Keyword(s):

Escherichia Coli ◽

Steady State ◽

Kinetic Analysis ◽

Atp Hydrolysis ◽

Reca Protein ◽

Steady State Kinetic ◽

Nucleoside Triphosphates ◽

Hydrolysis Of ◽

State Kinetic

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Fluorescent Probes for Live Cell Thiol Detection

Molecules ◽

10.3390/molecules26123575 ◽

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.

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Intracellular Ionic Strength Sensing Using NanoLuc

International Journal of Molecular Sciences ◽

10.3390/ijms22020677 ◽

2021 ◽

Vol 22 (2) ◽

pp. 677

Author(s):

Tausif Altamash ◽

Wesam Ahmed ◽

Saad Rasool ◽

Kabir H. Biswas

Keyword(s):

Thermal Stability ◽

Phase Separation ◽

Drug Discovery ◽

Ionic Strength ◽

Cellular Signaling ◽

Live Cells ◽

Protein Activity ◽

Cellular Survival ◽

Cellular Processes ◽

Wide Range

Intracellular ionic strength regulates myriad cellular processes that are fundamental to cellular survival and proliferation, including protein activity, aggregation, phase separation, and cell volume. It could be altered by changes in the activity of cellular signaling pathways, such as those that impact the activity of membrane-localized ion channels or by alterations in the microenvironmental osmolarity. Therefore, there is a demand for the development of sensitive tools for real-time monitoring of intracellular ionic strength. Here, we developed a bioluminescence-based intracellular ionic strength sensing strategy using the Nano Luciferase (NanoLuc) protein that has gained tremendous utility due to its high, long-lived bioluminescence output and thermal stability. Biochemical experiments using a recombinantly purified protein showed that NanoLuc bioluminescence is dependent on the ionic strength of the reaction buffer for a wide range of ionic strength conditions. Importantly, the decrease in the NanoLuc activity observed at higher ionic strengths could be reversed by decreasing the ionic strength of the reaction, thus making it suitable for sensing intracellular ionic strength alterations. Finally, we used an mNeonGreen–NanoLuc fusion protein to successfully monitor ionic strength alterations in a ratiometric manner through independent fluorescence and bioluminescence measurements in cell lysates and live cells. We envisage that the biosensing strategy developed here for detecting alterations in intracellular ionic strength will be applicable in a wide range of experiments, including high throughput cellular signaling, ion channel functional genomics, and drug discovery.

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

New Journal of Chemistry ◽

10.1039/c5nj03168k ◽

2016 ◽

Vol 40 (7) ◽

pp. 6101-6108 ◽

Cited By ~ 14

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.

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The AAA+ ATPase p97, a cellular multitool

Biochemical Journal ◽

10.1042/bcj20160783 ◽

2017 ◽

Vol 474 (17) ◽

pp. 2953-2976 ◽

Cited By ~ 42

Author(s):

Lasse Stach ◽

Paul S. Freemont

Keyword(s):

Atp Hydrolysis ◽

Current Status ◽

Cellular Functions ◽

Aaa Atpase ◽

Cellular Processes ◽

Proteotoxic Stress ◽

Binding Domains ◽

Wide Range ◽

Aaa Atpases ◽

Substrate Proteins

The AAA+ (ATPases associated with diverse cellular activities) ATPase p97 is essential to a wide range of cellular functions, including endoplasmic reticulum-associated degradation, membrane fusion, NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) activation and chromatin-associated processes, which are regulated by ubiquitination. p97 acts downstream from ubiquitin signaling events and utilizes the energy from ATP hydrolysis to extract its substrate proteins from cellular structures or multiprotein complexes. A multitude of p97 cofactors have evolved which are essential to p97 function. Ubiquitin-interacting domains and p97-binding domains combine to form bi-functional cofactors, whose complexes with p97 enable the enzyme to interact with a wide range of ubiquitinated substrates. A set of mutations in p97 have been shown to cause the multisystem proteinopathy inclusion body myopathy associated with Paget's disease of bone and frontotemporal dementia. In addition, p97 inhibition has been identified as a promising approach to provoke proteotoxic stress in tumors. In this review, we will describe the cellular processes governed by p97, how the cofactors interact with both p97 and its ubiquitinated substrates, p97 enzymology and the current status in developing p97 inhibitors for cancer therapy.

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