scholarly journals FLIM-Based Intracellular and Extracellular pH Measurements Using Genetically Encoded pH Sensor

Biosensors ◽  
2021 ◽  
Vol 11 (9) ◽  
pp. 340
Author(s):  
Alexander S. Goryashchenko ◽  
Alexey A. Pakhomov ◽  
Anastasia V. Ryabova ◽  
Igor D. Romanishkin ◽  
Eugene G. Maksimov ◽  
...  
Keyword(s):  
Cell Biology ◽  
Cytoplasmic Membrane ◽  
Transmembrane Domain ◽  
Fluorescent Sensor ◽  
Ph Sensor ◽  
Extracellular Ph ◽  
Live Cells ◽  
Ph Measurements ◽  
Quantitative Measurements

The determination of pH in live cells and tissues is of high importance in physiology and cell biology. In this report, we outline the process of the creation of SypHerExtra, a genetically encoded fluorescent sensor that is capable of measuring extracellular media pH in a mildly alkaline range. SypHerExtra is a protein created by fusing the previously described pH sensor SypHer3s with the neurexin transmembrane domain that targets its expression to the cytoplasmic membrane. We showed that with excitation at 445 nm, the fluorescence lifetime of both SypHer3s and SypHerExtra strongly depend on pH. Using FLIM microscopy in live eukaryotic cells, we demonstrated that SypHerExtra can be successfully used to determine extracellular pH, while SypHer3s can be applied to measure intracellular pH. Thus, these two sensors are suitable for quantitative measurements using the FLIM method, to determine intracellular and extracellular pH in a range from pH 7.5 to 9.5 in different biological systems.

scholarly journals Litmus-Body: a Molecularly Targeted Sensor for Cell-Surface pH Measurements

2019 ◽  
Author(s):  
Marc C. Goudge ◽  
Joe Chin-Hun Kuo ◽  
Ann E. Metzloff ◽  
Ling-Ting Huang ◽  
Marshall J. Colville ◽  
...  
Keyword(s):  
Cell Surface ◽  
Fluorescent Protein ◽  
Stokes Shift ◽  
Ph Sensor ◽  
Endocytic Pathway ◽  
Live Cells ◽  
Ph Measurements ◽  
Ph Changes ◽  
Specific Proteins ◽  
Green Fluorescent

ABSTRACTPrecise pH measurements in the immediate environment of receptors is essential for elucidating the mechanisms through which local pH changes associated with diseased phenotypes manifest into aberrant receptor function. However, current pH sensors lack the molecular specificity required to make these measurements. Herein we present the Litmus-body, our recombinant protein-based pH sensor, which through fusion to an anti-mouse IgG nanobody is capable of molecular targeting to specific proteins on the cell surface. By normalizing a pH-dependent green fluorescent protein to a long-Stokes shift red fluorophore or fluorescent protein, we readily report pH independent of sensor concentration using a single 488-nm excitation. Our Litmus-body showed excellent responsiveness in solution, with a greater than 50-fold change across the physiological regime of pH. The sensor was further validated for use on live cells, shown to be specific to the protein of interest, and was able to successfully recapitulate the numerous pH changes along the endocytic pathway.

scholarly journals Probing extracellular acidity of live cells in real time for cancer detection and monitoring anti-cancer drug activity

2015 ◽  
Vol 51 (32) ◽  
pp. 7015-7018 ◽  
Author(s):  
Bhawana Thakur ◽  
S. Jayakumar ◽  
Shilpa N. Sawant
Keyword(s):  
Cancer Cells ◽  
Real Time ◽  
Cancer Detection ◽  
Extracellular Ph ◽  
Live Cells ◽  
Cancer Drug ◽  
Drug Activity ◽  
Anti Cancer ◽  
Multifunctional Platform

A multifunctional platform is presented which (a) allows determination of extracellular pH in real time, (b) detects cancer cells, down to 5 cells, and (c) enables evaluating the efficacy of glycolysis inhibiting drugs.

scholarly journals Charged Residues Flanking the Transmembrane Domain of Two Related Toxin–Antitoxin System Toxins Affect Host Response

Toxins ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 329
Author(s):  
Andrew Holmes ◽  
Jessie Sadlon ◽  
Keith Weaver
Keyword(s):  
Amino Acid ◽  
Enterococcus Faecalis ◽  
Host Response ◽  
Cytoplasmic Membrane ◽  
Transmembrane Domain ◽  
The Other ◽  
Type I ◽  
Transcriptomic Response ◽  
Specific Amino Acid ◽  
Transporter Protein

A majority of toxins produced by type I toxin–antitoxin (TA-1) systems are small membrane-localized proteins that were initially proposed to kill cells by forming non-specific pores in the cytoplasmic membrane. The examination of the effects of numerous TA-1 systems indicates that this is not the mechanism of action of many of these proteins. Enterococcus faecalis produces two toxins of the Fst/Ldr family, one encoded on pheromone-responsive conjugative plasmids (FstpAD1) and the other on the chromosome, FstEF0409. Previous results demonstrated that overexpression of the toxins produced a differential transcriptomic response in E. faecalis cells. In this report, we identify the specific amino acid differences between the two toxins responsible for the differential response of a gene highly induced by FstpAD1 but not FstEF0409. In addition, we demonstrate that a transporter protein that is genetically linked to the chromosomal version of the TA-1 system functions to limit the toxicity of the protein.

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.

scholarly journals KD determination from time-resolved experiments on live cells with LigandTracer and reconciliation with end-point flow cytometry measurements

2021 ◽  
Author(s):  
Diana Spiegelberg ◽  
Jonas Stenberg ◽  
Pascale Richalet ◽  
Marc Vanhove
Keyword(s):  
Flow Cytometry ◽  
Live Cells ◽  
Time Resolved ◽  
Surface Receptors ◽  
Full Characterization ◽  
End Point ◽  
Titration Curves ◽  
Kinetics Of

AbstractDesign of next-generation therapeutics comes with new challenges and emulates technology and methods to meet them. Characterizing the binding of either natural ligands or therapeutic proteins to cell-surface receptors, for which relevant recombinant versions may not exist, represents one of these challenges. Here we report the characterization of the interaction of five different antibody therapeutics (Trastuzumab, Rituximab, Panitumumab, Pertuzumab, and Cetuximab) with their cognate target receptors using LigandTracer. The method offers the advantage of being performed on live cells, alleviating the need for a recombinant source of the receptor. Furthermore, time-resolved measurements, in addition to allowing the determination of the affinity of the studied drug to its target, give access to the binding kinetics thereby providing a full characterization of the system. In this study, we also compared time-resolved LigandTracer data with end-point KD determination from flow cytometry experiments and hypothesize that discrepancies between these two approaches, when they exist, generally come from flow cytometry titration curves being acquired prior to full equilibration of the system. Our data, however, show that knowledge of the kinetics of the interaction allows to reconcile the data obtained by flow cytometry and LigandTracer and demonstrate the complementarity of these two methods.

High-throughput extracellular pH monitoring and antibiotics screening by polymeric fluorescent sensor with LCST property

Methods ◽  
2019 ◽  
Vol 168 ◽  
pp. 51-61 ◽  
Author(s):  
Tingting Pan ◽  
Cheng Yang ◽  
Jiaze Li ◽  
Jiapei Jiang ◽  
Jiaxing Wen ◽  
...  
Keyword(s):  
High Throughput ◽  
Fluorescent Sensor ◽  
Ph Monitoring ◽  
Extracellular Ph

A cyanine-based fluorescent sensor for detecting endogenous zinc ions in live cells and organisms

Biomaterials ◽  
2012 ◽  
Vol 33 (31) ◽  
pp. 7818-7827 ◽  
Author(s):  
Zhiqian Guo ◽  
Gun-Hee Kim ◽  
Injae Shin ◽  
Juyoung Yoon
Keyword(s):  
Fluorescent Sensor ◽  
Live Cells ◽  
Zinc Ions

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.

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