cAMP Signaling: Methods and Protocols (Methods in Molecular Biology, 2483)

✍ Scribed by Manuela Zaccolo (editor)

• Publisher: Humana
• Year: 2022
• Tongue: English
• Leaves: 373
• Category: Library

✦ Synopsis

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 learning more about this important and developing field.

Chapter 7 is available open access under a Creative Commons Attribution 4.0 International License via link.springer.com.

✦ Table of Contents

Preface
Contents
Contributors
Chapter 1: Real-Time Measurements of Intracellular cAMP Gradients Using FRET-Based cAMP Nanorulers
1 Introduction
2 Materials
2.1 Cell Culture, Seeding, and Transfection
2.2 cDNA
2.3 Ligand Solution and Buffer Preparation
2.4 Single-Cell FRET Experiment
3 Methods
3.1 Cell Culture, Seeding, and Transfection
3.2 Mounting on Microscope
3.3 Focusing
3.4 ROI Selection
3.5 FRET Experiment
3.6 Data Analysis
4 Notes
References
Chapter 2: Assaying Protein Kinase A Activity Using a FRET-Based Sensor Purified from Mammalian Cells
1 Introduction
2 Materials
2.1 Mammalian Cell Expression
2.2 Reporter Purification
2.3 Ratiometric Plate Reader Assays
3 Methods
3.1 Optimizing Reporter Expression by PEI Transfection
3.2 Reporter Purification Following Scaled up Expression in HEK293T Cells
3.3 Calibrating the Reporter for Plate Reader Activity Assays
3.4 Determining the IC50 of a PKA Inhibitor
4 Notes
References
Chapter 3: MultiFRET: A Detailed Protocol for High-Throughput Multiplexed Ratiometric FRET
1 Introduction
2 Materials
3 Methods
3.1 Icy Setup
3.2 Initial Setup of MultiFRET
3.2.1 Active Contours
3.2.2 Excel Output Template (Optional)
3.2.3 Custom Calculations (Optional)
3.2.4 Sample Preparation (Example)
3.3 Running MultiFRET
4 Notes
References
Chapter 4: Photoactivated Adenylyl Cyclases as Optogenetic Modulators of Neuronal Activity
1 Introduction
2 Materials
2.1 Common Materials
2.2 C. elegans Strains
2.3 C. elegans Cultivation
2.4 Analyses of Behavior in Liquid
2.5 Analyses of Behavior on Solid Substrate
2.5.1 Body Length Measurements
2.5.2 Analyses of Crawling Behavior
2.6 cNMP Measurements
2.6.1 General Equipment and Materials
2.6.2 cAMP Measurements
2.6.3 cGMP Measurements
3 Methods
3.1 Optogenetic Tool Expression
3.2 C. elegans Cultivation
3.3 Analyses of Behavior in Liquid
3.4 Analyses of Behavior on Solid Substrate
3.4.1 Body Length Measurement
3.4.2 Analysis of Crawling Behavior
3.5 cNMP Measurements
3.5.1 C. elegans Extract Preparation
3.5.2 cAMP Measurements
3.5.3 The Subsequent Steps Should Be Carried out in the Dark
3.5.4 cGMP Measurements
4 Notes
References
Chapter 5: Imaging the cAMP Signaling Microdomain of the Primary Cilium Using Targeted FRET-Based Biosensors
1 Introduction
2 Materials
2.1 Stock Solutions
2.2 Materials
2.3 Working Solutions
2.4 Cell Culture Media
2.5 Microscope
3 Methods
3.1 Transfection: ``Drop Method´´
3.2 Serum Starving
3.3 FRET Measurements
3.4 Data Analysis
4 Caveats and Conclusions
5 Notes
References
Chapter 6: Methods to Assess Phosphodiesterase and/or Adenylyl Cyclase Activity Via Heterologous Expression in Fission Yeast
1 Introduction
2 Materials
2.1 Media
2.2 Chemicals for Mass Spectrometry Studies
2.2.1 Chemicals and Standards
2.2.2 Chromatography Mobile Phases
2.3 Equipment
2.3.1 Equipment for 5FOA Assays
2.3.2 Equipment for LC-MS/MS Measurements
3 Methods
3.1 5FOA Assays to Assess PDE Inhibitors
3.1.1 Strains that Lack AC Activity
3.1.2 Strains that Express a Functional AC Gene
3.2 Direct Measurement of Cyclic Nucleotide Levels for Assessing AC Inhibitors
3.2.1 Preparation of cAMP Samples
3.2.2 Measure Cyclic Nucleotide Levels Via Mass Spectrometry
4 Notes
References
Chapter 7: Time-Domain Fluorescence Lifetime Imaging of cAMP Levels with EPAC-Based FRET Sensors
1 Introduction
2 Materials
2.1 Stock Solutions
2.2 Disposables
2.3 Working Solutions
3 Methods
3.1 Microscope
3.2 Cell Culture
3.3 Transfection
3.4 Imaging
3.5 TSCPC FLIM
4 Notes
References
Chapter 8: Disruptors of AKAP-Dependent Protein-Protein Interactions
1 Introduction
2 Material
2.1 HTRF
2.2 AlphaScreen
2.3 Human Induced Pluripotent Stem Cells and Differentiation to Cardiac Myocytes
2.3.1 hiPSC Culture
2.3.2 hiPSC-CM Differentiation and Culture
2.3.3 Quality Control
2.3.4 Thawing of hiPSC-CMs
2.3.5 Calcium Imaging
2.3.6 Data Analysis
3 Methods
3.1 HTRF for Screening for Small Molecule Inhibitors of AKAP-PKA Interactions
3.1.1 Data Analysis
3.2 AlphaScreen Assay
3.2.1 Prepare Test Compound Dilution
3.2.2 Data Analysis
3.3 hiPSC Differentiation to Cardiac Myocytes
3.3.1 Geltrex Coating of 6-Well Cell culture Plates
3.3.2 hiPSC Culture
3.3.3 Cardiac Myocyte Differentiation
3.3.4 Quality Control
3.3.5 Calcium Imaging
3.3.6 Thawing of hiPSC-CMs
3.3.7 Calcium Imaging
3.3.8 Data Analysis
4 Notes
References
Chapter 9: Micro-2D Cell Culture for cAMP Measurements Using FRET Reporters in Human iPSC-Derived Cardiomyocytes
1 Introduction
2 Materials
2.1 Cell Culture Equipment
2.2 Consumable Supplies
2.3 FRET Equipment
2.4 Consumable Supplies for FRET Measurements
2.5 Analysis Equipment
3 Methods
3.1 Cell Seeding Numbers and Media Requirements for Different Cell Culture Vessel Formats
3.2 Preparation of Rings
3.3 Testing the Ring-Coverslip Assemblies for Leakage
3.4 Sterilizing the Ring-Coverslip Assemblies
3.5 Coating the Bottom Area Within the Rings
3.6 Seeding hIPS-Derived Cardiomyocytes into Rings
3.7 Maintaining hIPS-Derived Cardiomyocytes
3.8 Infection of Human IPS-Derived Cardiomyocytes for cAMP FRET Measurements
3.9 Transfection of hIPS-Derived Cardiomyocytes for cAMP FRET Measurements
3.10 cAMP FRET Measurements of hIPS-Derived Cardiomyocytes (See Note 36)
3.11 Analysis of cAMP FRET Measurements of hIPS-Derived Cardiomyocytes
4 Notes
References
Chapter 10: Automated Image Analysis of FRET Signals for Subcellular cAMP Quantification
1 Introduction
2 Materials
2.1 FRET-Based cAMP Measurement Components
2.2 Image Analysis Components
3 Methods
3.1 Image Acquisition: Spectral Imaging Fluorescence Microscopy of FRET-Based cAMP Reporters
3.2 Image Segmentation: Automated Whole-Cell cAMP Measurements
3.3 Feature Extraction: Quantitative Measurement of Whole-Cell Data
3.4 Automated Subcellular cAMP Measurements
3.5 Feature Extraction: Quantitative Subcellular Measurements
4 Notes
References
Chapter 11: In Vivo cAMP Dynamics in Drosophila Larval Neurons
1 Introduction
1.1 Epac 1-Camps Based FRET Biosensor
1.2 Anatomy of a Third Instar Larva
1.3 Generation of Transgenic Flies Using the GAL4-UAS System
1.4 Power and Caveats
2 Materials
2.1 Dissection
2.2 FRET Measurements
3 Methods
3.1 Generation of Transgenic Larvae Expressing Epac1-Camps Sensor in Neurons
3.2 Preparing the Dissecting Surface
3.3 Select Male Wandering Third Instar Larva
3.4 Dissection of Larva
3.5 FRET Measurements
3.6 Data Analysis
4 Notes
References
Chapter 12: Live Cell Imaging of Cyclic Nucleotides in Human Cardiomyocytes
1 Introduction
2 Materials
2.1 Isolation and Culture
2.2 Biosensors
2.3 Myocytes Isolation Solutions
2.4 Myocytes Culture and Transduction
2.5 FRET System
3 Methods
3.1 Obtaining and Transporting Human Tissue
3.2 Myocytes Isolation Prearrangements
3.3 Myocytes Isolation
3.4 Myocytes Culture
3.5 FRET Experiments
4 Notes
References
Chapter 13: Optogenetic Control of Heart Rhythm: Lightly Guiding the Cardiac Pace
1 Introduction
1.1 Overview
1.2 Physiology of Heart Rhythm Automaticity
1.3 Neurogenic Modulation of Heart Rate: Role of the Autonomic Nervous System
1.3.1 Cardiac Autonomic Innervation
1.3.2 Neurogenic Modulation of Cardiomyocyte Activity
1.4 Fundamentals of Optogenetics
1.5 Theoretical Bases of Cardiac Optogenetics
1.5.1 Advantages and Differences of Optogenetic vs. Conventional Cardiac Pacing Studies
1.5.2 Limitations of Cardiac Optogenetics
1.6 Model Systems for Cardiac Optogenetics
1.7 Optogenetic Control of Heart Rhythm
1.7.1 In Vitro Testing of the Function of the Chosen Opsin
1.7.2 Cell Selective Expression of ChR2 in the Mouse
2 Materials: Hardware Required to Perform In Vivo Cardiac Optogenetics
3 Methods
3.1 Preparation of the Mouse for In Vivo SN Optogenetics and Protocol of Photostimulation
3.2 Preparation of the Mouse and Optogenetic Protocol to Study Cardiac Electrophysiology
3.3 Data Collection and Analysis
4 Notes
References
Chapter 14: Live Imaging of cAMP Signaling in D. discoideum Based on a Bioluminescent Indicator, Nano-lantern (cAMP)
1 Introduction
2 Materials
2.1 CTZ Stock
2.2 D. discoideum Cells
2.3 CTZ for Bioluminescence Observation
3 Methods
3.1 Imaging of Cellular cAMP Dynamics in Buffer-Submerged Condition
3.2 Bioluminescence Imaging of the Spontaneous cAMP Signaling in Developing D. discoideum Cells on Agar Plate
3.2.1 Preparation of Agar Plate
3.2.2 Mounting Cells on Agar Plate
3.2.3 Bioluminescence Imaging of the Spontaneous cAMP Signaling
3.3 Spectroscopic Measurement
4 Notes
References
Chapter 15: Generation of Transgenic Mice Expressing Cytosolic and Targeted FRET Biosensors for cAMP and cGMP
1 Introduction
2 Materials
2.1 Equipment
2.2 Mice
2.3 Vectors
2.4 Cells
2.5 Substances and Solutions
2.6 Kits
2.7 Primers
2.8 LoxP Stop Sequence
2.9 Restriction Enzymes
2.10 Other Materials for DNA Purification
3 Methods
3.1 Cloning Strategy
3.2 PCR and DNA Extraction
3.3 Restriction Digest and Ligation
3.4 Transformation
3.5 Control Digest
3.6 Amplification of the Constructed Vector
3.7 Preparation of the DNA for the Microinjection
3.8 Genotyping
4 Notes
References
Chapter 16: How to Make the CUTiest Sensor in Three Simple Steps for Computational Pedestrians
1 Introduction
1.1 Beauty Is in the Eye of the Beholder
2 Methodology: Designing the CUTiest Sensor in Three Simple Steps
3 Experimental Validation
4 Conclusions
5 Notes
References
Chapter 17: Ion Channel-Based Reporters for cAMP Detection
1 Introduction
2 Materials
2.1 Fluorometric cAMP Measurements
2.2 Reagents
2.3 Electrophysiological cAMP Measurements
2.4 Data Analysis Software
3 Methods
3.1 cAMP Measurements Using a Spectrofluorometer
3.2 cAMP Measurements Using Electrophysiological Approaches
4 Notes
References
Chapter 18: Quantitative Phosphoproteomics to Study cAMP Signaling
1 Introduction
2 Materials and Reagents
2.1 Materials and Equipment
2.2 Working Solutions
2.2.1 For Cell Lysis
2.2.2 For Filter-Aided Sample Preparation (FASP)
2.2.3 For On-Column Stable Isotope Dimethyl Labeling
2.2.4 For Titanium Dioxide (TiO2)-Mediated Phosphopeptide Enrichment
2.2.5 For Tandem Mass Spectrometry (LC-MS/MS) Analysis
3 Methods
3.1 Sample Collection
3.2 Cell Lysis
3.3 FASP Digest
3.4 On-column Stable Isotope Dimethyl Labeling
3.5 Titanium Dioxide Phosphopeptide Enrichment
3.6 Mass Spectrometry Analysis
3.7 Quantitative Data Analysis
3.7.1 Peptide Searches
3.7.2 Downstream Data Processing and Statistical Analysis
4 Notes
4.1 Sample Collection
4.2 Cell Lysis
4.3 FASP Digest
4.4 Dimethyl Labeling
4.5 Phosphopeptide Enrichment
4.6 Mass Spectrometry
4.7 Quantitative Data Analysis
References
Chapter 19: Biochemical Analysis of AKAP-Anchored PKA Signaling Complexes
1 Introduction
1.1 RII-Selective Disruptors of AKAP Complexes
1.2 cAMP-Modulatory Effectors
1.3 PKA Inhibitors: From Proteins to Small Molecules
1.4 Chemical Inhibitors of PKA-C
2 Materials
2.1 Expression and Purification of AKAP-79, PKA-C, and PKA-RII Proteins
2.1.1 List of Required Chemicals and Reagents
2.2 In Vitro AKAP-RII-C Binding Assay
2.2.1 List of Reagents
3 Methods
3.1 Expression and Purification of Recombinant RII, C and AKAP Proteins in BL21 (DE3) pLysS E. coli
3.2 Lysis of the Bacterial Cell Pellet
3.3 Protein Purification
3.4 AKAP-RII Binding Assay (Fig. 3a)
3.5 SDS-PAGE and Coomassie Blue Staining
3.6 Biophysical Analysis of Purified Recombinant PKA Signaling Components
4 Notes
References
Chapter 20: Fluorescent Translocation Reporters for Sub-plasma Membrane cAMP Imaging
1 Introduction
2 Materials
2.1 Cell Culture and Transfection
2.2 Fluorescence Microscopy
3 Methods
3.1 Preparation of Poly-l-lysine-Coated Coverslips
3.2 Transfection of Cells
3.3 Time-Lapse Imaging of Sub-plasma Membrane cAMP Dynamics
3.4 Simultaneous Imaging of Sub-plasma Membrane Ca2+ and cAMP
3.5 Simultaneous Imaging of cAMP and PKA Activity
3.6 Simultaneous Imaging of cAMP and Subcellular Localization of Epac2
4 Notes
References
Chapter 21: A Live-Cell Imaging Assay for Nuclear Entry of cAMP-Dependent Protein Kinase Catalytic Subunits Stimulated by Endo...
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Imaging System
3 Methods
3.1 Cell Culture and Transient Transfection
3.2 Imaging
3.3 Data Analysis
4 Notes
References
Chapter 22: Measuring Spatiotemporal cAMP Dynamics Within an Endogenous Signaling Compartment Using FluoSTEP-ICUE
1 Introduction
1.1 cAMP/PKA Signaling
1.2 FRET-Based cAMP Reporters
1.3 Visualizing Local cAMP Signaling Dynamics
2 Materials
2.1 Stock Solutions (See Note 1)
2.2 Cell Culture and Transfection
2.3 Epifluorescence Microscope
2.4 Image Analysis
3 Methods
3.1 Cell Culture
3.2 PolyJet Transfection (See Note 1)
3.3 Attaching GFP11 to a POI via CRISPR-Cas9
3.4 Measuring Spatiotemporal cAMP Dynamics at an Endogenous POI
3.5 Analyzing FluoSTEP-ICUE FRET Data
4 Notes
References
Index