This volume discusses the latest techniques used by researchers to measure cAMP activity at the cell population, whole cell, and subcellular level. The techniques covered in the book address questions related to cAMP compartmentalization, which look at relevant protein-protein interactions that increase the spatial and temporal resolution of cAMP signals detection, and that can help in the integration of the increasingly complex information that is becoming available in this field. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.
Cutting-edge and comprehensive,
cAMP Signaling: Methods and Protocols, Second Edition is a valuable resource for scientists and researchers who are interested in lear
Table of ContentsPreface…
Table of Contents...
Contributing Authors…
1. Real-Time Measurements of Intracellular cAMP Gradients using FRET-Based cAMP Nanorulers
Charlotte Konrad, Martin J. Lohse, and Andreas Bock
2. Assaying Protein Kinase A Activity using a FRET-Based Sensor Purified from Mammalian Cells
Ashton J. Curtis, Ryan S. Dowsell, and Matthew G. Gold
3. MultiFRET: A Detailed Protocol for High-Throughput Multiplexed Ratiometric FRET
Masoud Ramuz, Ivan Diakonov, Chris Dunsby, and Julia Gorelik
4. Photoactivated Adenylyl Cyclases as Optogenetic Modulators of Neuronal Activity
Thilo Henss, Martin Schneider, Dennis Vettkötter, Wagner Steuer Costa, Jana F. Liewald, and Alexander Gottschalk
5. Imaging the cAMP Signaling Microdomain of the Primary Cilium using Targeted FRET-Based Biosensors
Danielle T. Arena and Aldebaran M. Hofer
6. Methods to Assess Phosphodiesterase and/or Adenylyl Cyclase Activity via Heterologous Expression in Fission Yeast
Marek Domin and Charles S. Hoffman
7. Time-Domain Fluorescence Lifetime Imaging of cAMP Levels with EPAC-Based FRET Sensors
Olga Kukk, Jeffrey Klarenbeek, and Kees Jalink
8. Disruptors of AKAP-Dependent Protein-Protein Interactions
Ryan Walker-Gray, Tamara Pallien, Duncan C. Miller, Andreas Oder, Martin Neuenschwander, Jens Peter von Kries, Sebastian Diecke, and Enno Klussmann
9. Micro-2D Cell Culture for cAMP Measurements using FRET Reporters in Human iPSC-Derived Cardiomyocytes
Andreas Koschinski and Manuela Zaccolo
10. Automated Image Analysis of FRET Signals for Subcellular cAMP Quantification
Silas J. Leavesley, Naga Annamdevula, Santina Johnson, DJ Pleshinger, and Thomas C. Rich
11. In Vivo cAMP Dynamics in Drosophila Larval Neurons
Isabella Maiellaro
12. Live Cell Imaging of Cyclic Nucleotides in Human Cardiomyocytes
Kira Beneke and Cristina E. Molina
13. Optogenetic Control of Heart Rhythm: Lightly Guiding the Cardiac Pace
Lolita Dokshokova, Nicola Pianca, Tania Zaglia, and Marco Mongillo
14. Live Imaging of cAMP Signaling in D. discoideum Based on a Bright Bioluminescent Protein, Nano-Lantern
Kazuki Horikawa and Takeharu Nagai
15. Generation of Transgenic Mice Expressing Cytosolic and Targeted FRET Biosensors for cAMP and cGMP
Roberta Kurelić and Viacheslav O. Nikolaev
16. How to Make the CUTiest Sensor in 3 Simple Steps for Computational Pedestrians
Florencia Klein, Cecilia Abreu, and Sergio Pantano
17. Ion-Channel Based Reporters for cAMP Detection
Thomas C. Rich, Wenkuan Xin, Silas J. Leavesley, C. Michael Francis, and Mark Taylor
18. Quantitative Phosphoproteomics to Study cAMP Signalling
Katharina Schleicher, Svenja Hester, Monika Stegmann, and Manuela Zaccolo
19. Biochemical Analysis of AKAP-Anchored PKA Signaling Complexes
Dominic P. Byrne, Mitchell H. Omar, Eileen J. Kennedy, Patrick A. Eyers, and John D. Scott
20. Fluorescent Translocation Reporters for Sub-Plasma Membrane cAMP Imaging
Oleg Dyachok, Yunjian Xu, Olof Idevall-Hagren, and Anders Tengholm
21. A Live-Cell Imaging Assay for Nuclear Entry of cAMP-Dependent Protein Kinase Catalytic Subunits Stimulated by Endogenous GPCR Activation
Grace E. Peng and Mark von Zastrow
22. Measuring Spatiotemporal cAMP Dynamics within an Endogenous Signaling Compartment using FluoSTEP-ICUE
Julia C. Hardy, Sohum Mehta, and Jin Zhang
Subject Index List…
