Use of single-cell imaging techniques to assess the regulation of cAMP dynamics

2006 ◽  
Vol 34 (4) ◽  
pp. 468-471 ◽  
Author(s):  
D. Willoughby ◽  
D.M.F. Cooper
Keyword(s):  
Single Cell ◽  
Real Time ◽  
Imaging Techniques ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Cell System ◽  
Cellular Processes ◽  
Signalling Molecule ◽  
Anchoring Protein ◽  
Model Cell

cAMP is a ubiquitous intracellular signalling molecule that can regulate a wide array of cellular processes. The diversity of action of this second messenger owes much to the localized generation, action and hydrolysis of cAMP within discrete subcellular regions. Further signalling specificity can be achieved by the ability of cells to modulate the frequency or incidence of such cAMP signals. Here, we discuss the use of two cAMP biosensors that measure real-time cAMP changes in the single cell, to address the mechanisms underlying the generation of dynamic cAMP signals. The first method monitors sub-plasmalemmal cAMP changes using mutant cyclic nucleotide-gated channels and identifies an AKAP (A-kinase-anchoring protein)–protein kinase A–PDE4 (phosphodiesterase-4) signalling complex that is central to the generation of dynamic cAMP transients in this region of the cell. The second study uses a fluorescence resonance energy transfer-based cAMP probe, based on Epac1 (exchange protein directly activated by cAMP 1), to examine interplay between Ca2+ and cAMP signals. This study demonstrates real-time oscillations in cAMP driven by a Ca2+-stimulated AC (adenylate cyclase) (AC8) and subsequent PDE4 activity. These studies, using two very different single-cell cAMP probes, broaden our understanding of the specific spatiotemporal characteristics of agonist-evoked cAMP signals in a model cell system.

scholarly journals AIE-Driven Fluorescent Polysaccharide Polymersome as Enzyme-responsive FRET Nanoprobe to Study the Real-time Delivery Aspects in Live-Cells

2021 ◽  
Author(s):  
Nilesh Umakant Deshpande ◽  
Mishika Virmani ◽  
Manickam Jayakannan
Keyword(s):  
Energy Transfer ◽  
Confocal Microscopy ◽  
Real Time ◽  
Fluorescence Resonance Energy Transfer ◽  
Imaging Techniques ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Live Cells ◽  
Intracellular Enzyme ◽  
Bio Imaging

We report aggregation induced emission (AIE) driven polysaccharide polymersome as fluorescence resonance energy transfer (FRET) nanoprobes to study their intracellular enzyme-responsive delivery by real-time live-cell confocal microscopy bio-imaging techniques. AIE...

scholarly journals Genetically Encoded Biosensors Based on Fluorescent Proteins

Sensors ◽  
2021 ◽  
Vol 21 (3) ◽  
pp. 795
Author(s):  
Hyunbin Kim ◽  
Jeongmin Ju ◽  
Hae Nim Lee ◽  
Hyeyeon Chun ◽  
Jihye Seong
Keyword(s):  
Molecular Dynamics ◽  
Energy Transfer ◽  
Real Time ◽  
Fluorescent Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Critical Factors ◽  
Successful Development ◽  
Cellular Processes ◽  
Molecular Events

Genetically encoded biosensors based on fluorescent proteins (FPs) allow for the real-time monitoring of molecular dynamics in space and time, which are crucial for the proper functioning and regulation of complex cellular processes. Depending on the types of molecular events to be monitored, different sensing strategies need to be applied for the best design of FP-based biosensors. Here, we review genetically encoded biosensors based on FPs with various sensing strategies, for example, translocation, fluorescence resonance energy transfer (FRET), reconstitution of split FP, pH sensitivity, maturation speed, and so on. We introduce general principles of each sensing strategy and discuss critical factors to be considered if available, then provide representative examples of these FP-based biosensors. These will help in designing the best sensing strategy for the successful development of new genetically encoded biosensors based on FPs.

scholarly journals Designing and Development of FRET-Based Nanosensor for Real Time Analysis of N-Acetyl-5-Neuraminic Acid in Living Cells

2021 ◽  
Vol 8 ◽  
Author(s):  
Ruphi Naz ◽  
Mohammad K. Okla ◽  
Urooj Fatima ◽  
Mohd. Mohsin ◽  
Walid H. Soufan ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Metabolic Engineering ◽  
Sialic Acid ◽  
Real Time ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Neuraminic Acid ◽  
Yeast Cells ◽  
Metabolite Level ◽  
Health Maintenance

N-acetyl-5-neuraminic acid (NeuAc) plays crucial role in improving the growth, brain development, brain health maintenance, and immunity enhancement of infants. Commercially, it is used in the production of antiviral drugs, infant milk formulas, cosmetics, dietary supplements, and pharmaceutical products. Because of the rapidly increasing demand, metabolic engineering approach has attracted increasing attention for NeuAc biosynthesis. However, knowledge of metabolite flux in biosynthetic pathways is one of the major challenges in the practice of metabolic engineering. So, an understanding of the flux of NeuAc is needed to determine its cellular level at real time. The analysis of the flux can only be performed using a tool that has the capacity to measure metabolite level in cells without affecting other metabolic processes. A Fluorescence Resonance Energy Transfer (FRET)-based genetically-encoded nanosensor has been generated in this study to monitor the level of NeuAc in prokaryotic and eukaryotic cells. Sialic acid periplasmic binding protein (SiaP) from Haemophilus influenzae was exploited as a sensory element for the generation of nanosensor. The enhanced cyan fluorescent protein (ECFP) and Venus were used as Fluroscence Resonance Energy Transfer (FRET) pair. The nanosensor, which was termed fluorescent indicator protein for sialic acid (FLIP-SA), was successfully transformed into, and expressed in Escherichia coli BL21 (DE3) cells. The expressed protein of the nanosensor was isolated and purified. The purified nanosensor protein was characterized to assess the affinity, specificity, and stability in the pH range. The developed nanosensor exhibited FRET change after addition to NeuAc. The developed nanosensor was highly specific, exhibited pH stability, and detected NeuAc levels in the nanomolar to milimolar range. FLIP-SA was successfully introduced in bacterial and yeast cells and reported the real-time intracellular levels of NeuAc non-invasively. The FLIP-SA is an excellent tool for the metabolic flux analysis of the NeuAc biosynthetic pathway and, thus, may help unravel the regulatory mechanism of the metabolic pathway of NeuAc. Furthermore, FLIP-SA can be used for the high-throughput screening of E. coli mutant libraries for varied NeuAc production levels.

scholarly journals Genetically Encoded Fluorescent Sensor for Poly-ADP-Ribose

2020 ◽  
Vol 21 (14) ◽  
pp. 5004
Author(s):  
Ekaterina O. Serebrovskaya ◽  
Nadezda M. Podvalnaya ◽  
Varvara V. Dudenkova ◽  
Anna S. Efremova ◽  
Nadya G. Gurskaya ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Mammalian Cells ◽  
Fluorescent Proteins ◽  
Fluorescent Sensor ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Post Translational Modification ◽  
Cellular Processes ◽  
Hydrogen Peroxide Treatment ◽  
Damage Response

Poly-(ADP-ribosyl)-ation (PARylation) is a reversible post-translational modification of proteins and DNA that plays an important role in various cellular processes such as DNA damage response, replication, transcription, and cell death. Here we designed a fully genetically encoded fluorescent sensor for poly-(ADP-ribose) (PAR) based on Förster resonance energy transfer (FRET). The WWE domain, which recognizes iso-ADP-ribose internal PAR-specific structural unit, was used as a PAR-targeting module. The sensor consisted of cyan Turquoise2 and yellow Venus fluorescent proteins, each in fusion with the WWE domain of RNF146 E3 ubiquitin ligase protein. This bipartite sensor named sPARroW (sensor for PAR relying on WWE) enabled monitoring of PAR accumulation and depletion in live mammalian cells in response to different stimuli, namely hydrogen peroxide treatment, UV irradiation and hyperthermia.

scholarly journals Genotyping of Benzimidazole-Resistant and Dicarboximide-Resistant Mutations in Botrytis cinerea Using Real-Time Polymerase Chain Reaction Assays

2008 ◽  
Vol 98 (4) ◽  
pp. 397-404 ◽  
Author(s):  
Shinpei Banno ◽  
Fumiyasu Fukumori ◽  
Akihiko Ichiishi ◽  
Kiyotsugu Okada ◽  
Hidetoshi Uekusa ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Botrytis Cinerea ◽  
Melting Curve ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Cross Resistance ◽  
Melting Curve Analysis ◽  
Chain Reaction ◽  
Polymerase Chain

Botrytis cinerea, an economically important gray mold pathogen, frequently exhibits multiple fungicide resistance. A fluorescence resonance energy transfer-based real-time polymerase chain reaction assay has been developed to detect benzimidazole- and dicarboximide-resistant mutations. Three benzimidazole-resistant mutations—198Glu to Ala (E198A), F200Y, and E198K—in β-tubulin BenA were detected using a single set of fluorescence-labeled sensor and anchor probes by melting curve analysis. Similarly, three dicarboximide-resistant mutations—I365S, V368F plus Q369H, and Q369P—in the histidine kinase BcOS1 were successfully distinguished. Unassigned melting profiles in BenA genotyping assay resulted in the identification of a new benzimidazole-resistant BenA E198V mutation. This mutation conferred resistance to carbendazim as do E198A and E198K mutations. The isolates with BenA E198V mutation showed a negative cross-resistance to diethofencarb, but to a lesser extent than the E198A mutants. A survey of 210 B. cinerea field isolates revealed that most of benzimidazole-resistant isolates possessed the E198V or E198A mutation in the BenA gene, and the I365S mutation in the BcOS1 gene was also frequently observed in Japanese isolates. However, benzimidazole-resistant isolates with BenA F200Y or E198K mutations, which confer the diethofencarb-insensitive phenotype, were rare. Our BenA and BcOS1 genotyping is a rapid and reliable method that is suitable for monitoring the fungicide-resistant field population.

scholarly journals Imaging kinase–AKAP79–phosphatase scaffold complexes at the plasma membrane in living cells using FRET microscopy

2002 ◽  
Vol 160 (1) ◽  
pp. 101-112 ◽  
Author(s):  
Seth F. Oliveria ◽  
Lisa L. Gomez ◽  
Mark L. Dell'Acqua
Keyword(s):  
Signal Transduction ◽  
Glutamate Receptors ◽  
Direct Evidence ◽  
Protein A ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Adaptor Proteins ◽  
Living Cells ◽  
Intact Cells ◽  
Anchoring Protein

Scaffold, anchoring, and adaptor proteins coordinate the assembly and localization of signaling complexes providing efficiency and specificity in signal transduction. The PKA, PKC, and protein phosphatase-2B/calcineurin (CaN) scaffold protein A–kinase anchoring protein (AKAP) 79 is localized to excitatory neuronal synapses where it is recruited to glutamate receptors by interactions with membrane-associated guanylate kinase (MAGUK) scaffold proteins. Anchored PKA and CaN in these complexes could have important functions in regulating glutamate receptors in synaptic plasticity. However, direct evidence for the assembly of complexes containing PKA, CaN, AKAP79, and MAGUKs in intact cells has not been available. In this report, we use immunofluorescence and fluorescence resonance energy transfer (FRET) microscopy to demonstrate membrane cytoskeleton–localized assembly of this complex. Using FRET, we directly observed binding of CaN catalytic A subunit (CaNA) and PKA-RII subunits to membrane-targeted AKAP79. We also detected FRET between CaNA and PKA-RII bound simultaneously to AKAP79 within 50 Å of each other, thus providing the first direct evidence of a ternary kinase–scaffold–phosphatase complex in living cells. This finding of AKAP-mediated PKA and CaN colocalization on a nanometer scale gives new appreciation to the level of compartmentalized signal transduction possible within scaffolds. Finally, we demonstrated AKAP79-regulated membrane localization of the MAGUK synapse-associated protein 97 (SAP97), suggesting that AKAP79 functions to organize even larger signaling complexes.

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