scholarly journals A rationally designed c-di-AMP FRET biosensor to monitor nucleotide dynamics

2021 ◽  
Author(s):  
Alex J. Pollock ◽  
Philip H. Choi ◽  
Shivam A. Zaver ◽  
Liang Tong ◽  
Joshua J. Woodward
Keyword(s):  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Second Messenger ◽  
Adenosine Monophosphate ◽  
Luria Broth ◽  
Effector Proteins ◽  
Detection Methods ◽  
Live Cells ◽  
Bacterial Populations

3’3’-cyclic di-adenosine monophosphate (c-di-AMP) is an important nucleotide second messenger found throughout the bacterial domain of life. C-di-AMP is essential in many bacteria and regulates a diverse array of effector proteins controlling pathogenesis, cell wall homeostasis, osmoregulation, and central metabolism. Despite the ubiquity and importance of c-di-AMP, methods to detect this signaling molecule are limited, particularly at single cell resolution. In this work, crystallization of the Listeria monocytogenes c-di-AMP effector protein Lmo0553 enabled structure guided design of a Förster resonance energy transfer (FRET) based biosensor, which we have named CDA5. CDA5 is a fully genetically encodable, specific, and reversible biosensor which allows for the detection of c-di-AMP dynamics both in vitro and within live cells in a nondestructive manner. Our initial studies identify a distribution of c-di-AMP in Bacillus subtilis populations first grown in Luria Broth and then resuspended in diluted Luria Broth compatible with florescence analysis. Furthermore, we find that B. subtilis mutants lacking either a c-di-AMP phosphodiesterase or cyclase have respectively higher and lower FRET responses. These findings provide novel insight into the c-di-AMP distribution within bacterial populations and establish CDA5 as a powerful platform for characterizing new aspects of c-di-AMP regulation. Importance C-di-AMP is an important nucleotide second messenger for which detection methods are severely limited. In this work we engineer and implement a c-di-AMP specific FRET biosensor to remedy this dearth. We present this biosensor, CDA5, as a versatile tool to investigate previously intractable facets of c-di-AMP biology.

scholarly journals A rationally designed c-di-AMP FRET biosensor to monitor nucleotide dynamics

2021 ◽  
Author(s):  
Alex J. Pollock ◽  
Philip H. Choi ◽  
Shivam A. Zaver ◽  
Liang Tong ◽  
Joshua J. Woodward
Keyword(s):  
Single Cells ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Second Messenger ◽  
Adenosine Monophosphate ◽  
Luria Broth ◽  
Effector Proteins ◽  
Detection Methods ◽  
Bacterial Populations

ABSTRACT3’3’-cyclic di-adenosine monophosphate (c-di-AMP) is an important nucleotide second messenger found throughout the bacterial domain of life. C-di-AMP is essential in many bacteria and regulates a diverse array of effector proteins controlling pathogenesis, cell wall homeostasis, osmoregulation, and central metabolism. Despite the ubiquity and importance of c-di-AMP, methods to detect this signaling molecule are limited, particularly at single cell resolution. In this work, crystallization of the Listeria monocytogenes c-di-AMP effector protein Lmo0553 enabled structure guided design of a Förster resonance energy transfer (FRET) based biosensor, which we have named CDA5. CDA5 is a fully genetically encodable, specific, and reversible biosensor which allows for the detection of c-di-AMP dynamics both in vitro and within live single cells in a nondestructive manner. Our initial studies identify a unimodal distribution of c-di-AMP in Bacillus subtilis which decreases rapidly when cells are grown in diluted Luria Broth. Furthermore, we find that B. subtilis mutants lacking either a c-di-AMP phosphodiesterase or cyclase have respectively higher and lower FRET responses, again in a unimodal manner. These findings provide novel insight into c-di-AMP distribution within bacterial populations and establish CDA5 as a powerful platform for characterizing new aspects of c-di-AMP regulation.ImportanceC-di-AMP is an important nucleotide second messenger for which detection methods are severely limited. In this work we engineer and implement a c-di-AMP specific FRET biosensor to remedy this dearth. We present this biosensor, CDA5, as a versatile tool to investigate previously intractable facets of c-di-AMP biology.

Analisis of A-Kinase Anchoring Protein Interactions With PKA Using Fluorescence Resonance Energy Transfer

2000 ◽  
Vol 6 (S2) ◽  
pp. 828-829
Author(s):  
M. L. Ruehr ◽  
D. S. Damron ◽  
M. Bond
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Regulatory Subunit ◽  
Live Cells ◽  
Human Thyroid ◽  
Local Concentration ◽  
Fluorescence Resonance

The clustering of components of a signaling pathway at a specific subcellular location raises the local concentration of the appropriate messengers and serves to amplify the signal. The cAMP dependent-protein kinase (PKA) pathway is regulated by compartmentalization of its components. A-kinase anchoring proteins (AKAPs) tether PKA to specific subcellular sites, thus presumably increasing substrate specificity. Phosphorylation of the type II regulatory subunit of PKA (RII) increases its affinity for AKAPs in vitro (1). The purpose of this study was to investigate whether altering the phosphorylation state of RII in live cells changes its affinity for an AKAP. Specifically, we investigated the binding kinetics between Ht31, a peptide containing the PKA binding portion of an AKAP from human thyroid (2), and RII, in response to PKA activators or inhibitors.Fluorescence resonance energy transfer (FRET) was used to monitor binding events between RII and the catalytic subunit (C) of PKA, Ht31, or Ht31P, a mutated form of Ht31 which does not bind RII.

scholarly journals BacMam System for FRET-Based cAMP Sensor Expression in Studies of Melanocortin MC1 Receptor Activation

2012 ◽  
Vol 17 (8) ◽  
pp. 1096-1101 ◽  
Author(s):  
Olga Mazina ◽  
Reet Reinart-Okugbeni ◽  
Sergei Kopanchuk ◽  
Ago Rinken
Keyword(s):  
High Throughput Screening ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Cyclic Adenosine Monophosphate ◽  
Second Messenger ◽  
Adenosine Monophosphate ◽  
Receptor Activation ◽  
Dependent Manner ◽  
Endogenous Expression ◽  
Mammalian Cell Lines

Cyclic adenosine monophosphate (cAMP) is a second messenger of many G-protein-coupled receptors (GPCRs) and a useful readout molecule to estimate the biological activity of various GPCR-specific agents. Here we report the development and use of a Förster resonance energy transfer (FRET) biosensor for cAMP (Epac2-camps) combined with a baculovirus-based BacMam transduction system. The constructed BacMam-Epac2-camps viral transduction system is a simple and robust tool for ligand screening at the second-messenger level in a variety of mammalian cell lines. The level of biosensor protein expression can easily be adjusted in a dose-dependent manner depending on the multiplicity of viral infection. For setting up the assay, we used a B16F10 murine melanoma cell line with endogenous expression of melanocortin-1 receptor (MC1R). The receptor activation was characterized by a set of MC1R full and partial agonists. Bivalent ions Ca2+ as well as Mg2+ modulated ligand potencies, whereas the effect was ligand and ion specific. Results obtained for MC1R indicate that the BacMam-Epac2-camps system may also be applicable for studying the activation of other GPCRs and may be implemented in routine analysis as well as in high-throughput screening.

scholarly journals FluoSTEPs: Fluorescent biosensors for monitoring compartmentalized signaling within endogenous microdomains

2021 ◽  
Vol 7 (21) ◽  
pp. eabe4091
Author(s):  
Brian Tenner ◽  
Jason Z. Zhang ◽  
Yonghoon Kwon ◽  
Veronica Pessino ◽  
Siyu Feng ◽  
...  
Keyword(s):  
Intracellular Signaling ◽  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Regulatory Elements ◽  
Live Cells ◽  
Fluorescent Biosensors ◽  
Signaling Events

Growing evidence suggests that many essential intracellular signaling events are compartmentalized within kinetically distinct microdomains in cells. Genetically encoded fluorescent biosensors are powerful tools to dissect compartmentalized signaling, but current approaches to probe these microdomains typically rely on biosensor fusion and overexpression of critical regulatory elements. Here, we present a novel class of biosensors named FluoSTEPs (fluorescent sensors targeted to endogenous proteins) that combine self-complementing split green fluorescent protein, CRISPR-mediated knock-in, and fluorescence resonance energy transfer biosensor technology to probe compartmentalized signaling dynamics in situ. We designed FluoSTEPs for simultaneously highlighting endogenous microdomains and reporting domain-specific, real-time signaling events including kinase activities, guanosine triphosphatase activation, and second messenger dynamics in live cells. A FluoSTEP for 3′,5′-cyclic adenosine monophosphate (cAMP) revealed distinct cAMP dynamics within clathrin microdomains in response to stimulation of G protein–coupled receptors, showcasing the utility of FluoSTEPs in probing spatiotemporal regulation within endogenous signaling architectures.

scholarly journals Spatial and Temporal Regulation of Focal Adhesion Kinase Activity in Living Cells

2007 ◽  
Vol 28 (1) ◽  
pp. 201-214 ◽  
Author(s):  
Xinming Cai ◽  
Daniel Lietha ◽  
Derek F. Ceccarelli ◽  
Andrei V. Karginov ◽  
Zenon Rajfur ◽  
...  
Keyword(s):  
Focal Adhesion Kinase ◽  
Conformational Change ◽  
Focal Adhesion ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Living Cells ◽  
Live Cells ◽  
Major Mechanism

ABSTRACT Focal adhesion kinase (FAK) is an essential kinase that regulates developmental processes and functions in the pathology of human disease. An intramolecular autoinhibitory interaction between the FERM and catalytic domains is a major mechanism of regulation. Based upon structural studies, a fluorescence resonance energy transfer (FRET)-based FAK biosensor that discriminates between autoinhibited and active conformations of the kinase was developed. This biosensor was used to probe FAK conformational change in live cells and the mechanism of regulation. The biosensor demonstrates directly that FAK undergoes conformational change in vivo in response to activating stimuli. A conserved FERM domain basic patch is required for this conformational change and for interaction with a novel ligand for FAK, acidic phospholipids. Binding to phosphatidylinositol 4,5-bisphosphate (PIP2)-containing phospholipid vesicles activated and induced conformational change in FAK in vitro, and alteration of PIP2 levels in vivo changed the level of activation of the conformational biosensor. These findings provide direct evidence of conformational regulation of FAK in living cells and novel insight into the mechanism regulating FAK conformation.

scholarly journals Genetically encoded ratiometric indicators for potassium ion

2018 ◽  
Author(s):  
Yi Shen ◽  
Sheng-Yi Wu ◽  
Vladimir Rancic ◽  
Yong Qian ◽  
Shin-Ichiro Miyashita ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Temporal Dynamics ◽  
Biological Activities ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Potassium Ion ◽  
Live Cells ◽  
Fret Efficiency

AbstractPotassium ion (K+) homeostasis and dynamics play critical roles in regulating various biological activities, and the ability to monitor K+ spatial-temporal dynamics is critical to understanding these biological functions. Here we report the design and characterization of a Förster resonance energy transfer (FRET)-based genetically encoded K+ indicator, KIRIN1, constructed by inserting a bacterial cytosolic K+ binding protein (Kbp) between a fluorescent protein (FP) FRET pair, mCerulean3 and cp173Venus. Binding of K+ induces a conformational change in Kbp, resulting in an increase in FRET efficiency. KIRIN1 was able to detect K+ at physiologically relevant concentrations in vitro and is highly selective toward K+ over Na+. We further demonstrated that KIRIN1 allowed real-time imaging of pharmacologically induced depletion of cytosolic K+ in live cells, and KIRIN1 also enabled optical tracing of K+ efflux and reuptake in neurons upon glutamate stimulation in cultured primary neurons. These results demonstrate that KIRIN1 is a valuable tool to detect K+in vitro and in live cells.

scholarly journals In-Vitro Characterization of mCerulean3_mRuby3 as a Novel FRET Pair with Favorable Bleed-Through Characteristics

Biosensors ◽  
2019 ◽  
Vol 9 (1) ◽  
pp. 33 ◽  
Author(s):  
Kira Erismann-Ebner ◽  
Anne Marowsky ◽  
Michael Arand
Keyword(s):  
Interaction Analysis ◽  
Signal To Noise Ratio ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Live Cells ◽  
Promising Alternative ◽  
In Vitro Characterization ◽  
Bleed Through ◽  
Fret Pair

In previous studies, we encountered substantial problems using the CFP_YFP Förster resonance energy transfer (FRET) pair to analyze protein proximity in the endoplasmic reticulum of live cells. Bleed-through of the donor emission into the FRET channel and overlap of the FRET emission wavelength with highly variable cellular autofluorescence significantly compromised the sensitivity of our analyses. Here, we propose mCerulean3 and mRuby3 as a new FRET pair to potentially overcome these problems. Fusion of the two partners with a trypsin-cleavable linker allowed the direct comparison of the FRET signal characteristics of the associated partners with those of the completely dissociated partners. We compared our new FRET pair with the canonical CFP_YFP and the more recent mClover3_mRuby3 pairs and found that, despite a lower total FRET signal intensity, the novel pair had a significantly better signal to noise ratio due to lower donor emission bleed-through. This and the fact that the mRuby3 emission spectrum did not overlap with that of common cellular autofluorescence renders the mCerulean3_mRuby3 FRET pair a promising alternative to the common CFP_YFP FRET pair for the interaction analysis of membrane proteins in living cells.

Quantitative Ratiometric pH Imaging Using a 1,2-Dioxetane Chemiluminescence Resonance Energy Transfer Sensor

2020 ◽  
Author(s):  
Lucas S. Ryan ◽  
Jeni Gerberich ◽  
Uroob Haris ◽  
ralph mason ◽  
Alexander Lippert
Keyword(s):  
Energy Transfer ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Whole Body ◽  
Photon Flux ◽  
Ph Homeostasis ◽  
Ph Imaging ◽  
Confounding Variables ◽  
Significant Difference

<p>Regulation of physiological pH is integral for proper whole-body and cellular function, and disruptions in pH homeostasis can be both a cause and effect of disease. In light of this, many methods have been developed to monitor pH in cells and animals. In this study, we report a chemiluminescence resonance energy transfer (CRET) probe Ratio-pHCL-1, comprised of an acrylamide 1,2-dioxetane chemiluminescent scaffold with an appended pH-sensitive carbofluorescein fluorophore. The probe provides an accurate measurement of pH between 6.8-8.4, making it viable tool for measuring pH in biological systems. Further, its ratiometric output is independent of confounding variables. Quantification of pH can be accomplished both using common fluorimetry and advanced optical imaging methods. Using an IVIS Spectrum, pH can be quantified through tissue with Ratio-pHCL-1, which has been shown in vitro and precisely calibrated in sacrificed mouse models. Initial studies showed that intraperitoneal injections of Ratio-pHCL-1 into sacrificed mice produce a photon flux of more than 10^10 photons per second, and showed a significant difference in ratio of emission intensities between pH 6.0, 7.0, and 8.0.</p> <b></b><i></i><u></u><sub></sub><sup></sup><br>

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