The Integrin Interactome: Methods and Protocols 1071609610, 9781071609613

Table of contents :
Preface
Early Days: Purifying and Identifying Adhesive Receptors and Their Ligands
The Rise and Fall of Integrins as Therapeutic Targets
The Integrin Adhesome, Optical Super-Resolution, and Quantitative Microscopy
Force-Dependent Integrin Activation
Current Outlook and Future Perspectives
References
Acknowledgement
Contents
Contributors
Part I: Integrin Activation and Focal Adhesion Measurements
Chapter 1: Measurement of Integrin Activation and Conformational Changes on the Cell Surface by Soluble Ligand and Antibody Bi...
1 Introduction
2 Materials
2.1 DNA Constructs
2.2 Integrin Ligands
2.3 Preparation of FN9-10 Fragment
2.4 Preparation of ICAM-1-Fc Fragment
2.5 Integrin Antibodies
2.6 Secondary Antibodies and Other Probes
2.7 Integrin Inhibitors
2.8 Cell Culture Medium and Transfection
3 Methods
3.1 Protein Ligand Preparation
3.2 FN9-10 Fragment Preparation
3.3 ICAM-1-Fc Fragment Preparation
3.4 Protein Ligand and Antibody Labeling
3.5 Cell Transfection
3.6 Soluble Ligand and Antibody Binding Assay
3.7 Flow Cytometry
3.8 Data Analysis
3.9 An Alternative Method
4 Notes
References
Chapter 2: Quantification of Integrin Activation and Ligation in Adherent Cells
1 Introduction
2 Materials
2.1 α5β1 Activation Assay
2.1.1 Growing GST-FNIII9-11 Peptide
2.1.2 Purification of GST-FNIII9-11 Peptide
2.1.3 Activation Assay
2.1.4 Immunoblotting
2.2 αvβ3 Activation Assay
2.2.1 Activation Assay
2.2.2 Immunoblotting
2.3 Integrin Ligation Measurement
2.3.1 Western Blotting
2.4 Immunocytochemistry
3 Methods
3.1 α5β1 Activation Assay
3.1.1 Growing GST-FNIII9-11 Peptide
3.1.2 Purification of GST-FNIII9-11 Peptide
3.1.3 Activation Assay
3.1.4 Immunoblotting
3.2 αvβ3 Activation Assay
3.2.1 Activation Assay
3.2.2 Immunoblotting
3.3 Integrin Ligation Measurement
3.3.1 By Western Blot
3.3.2 By Immunocytochemistry
4 Notes
References
Chapter 3: Multiparametric Analysis of Focal Adhesions in Bidimensional Substrates
1 Introduction
2 Materials
2.1 Cell and Substrate Preparation
2.2 Fixation and Staining
2.3 Mounting and Image Collection
2.4 Image Analysis and Parameter Quantification
3 Methods
3.1 Coverslip Coating
3.2 Fibronectin Solution Preparation and Coverslip Coating
3.3 Coverslip Blocking
3.4 Cell Preparation and Adhesion
3.5 Fixation
3.6 Permeabilization
3.7 Blocking and Staining
3.8 Mounting
3.9 Image Collection
3.10 Image Analysis
3.11 Data Representation and Statistics
4 Notes
References
Chapter 4: Focal Adhesion Isolation Assay Using ECM-Coated Magnetic Beads
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Bead Functionalization and Coating
2.3 Bead/Cell Incubation and Force Application
2.4 Focal Adhesion Isolation
3 Methods
3.1 Bead Functionalization
3.2 Cell/Bead Incubation
3.3 Magnetics
3.4 Cross-Linking (Optional)
3.5 Cell Lysis and Bead Separation
4 Notes
References
Part II: Proximity and Microscopy-Based Methods to Determine Integrin Interactions
Chapter 5: Functional Integrin Regulation Through Interactions with Tetraspanin CD9
1 Introduction
2 Materials
2.1 Cell Cultures
2.2 Buffers and Reagents
2.3 Antibodies
2.4 Instruments and Software
3 Methods
3.1 Specific Coimmunoprecipitation of CD9 and Integrin Molecules from the Cell Surface
3.2 Covalent Chemical Cross-Linking
3.3 Pull-Down Assays
3.4 Proximity Ligation Assay
4 Notes
References
Chapter 6: Proximity-Dependent Biotinylation (BioID) of Integrin Interaction Partners
1 Introduction
2 Materials
2.1 Generation of Stable BirA* Expressing Cell Lines
2.2 Maturation of BirA Fusion Constructs
2.3 Localization of BirA Fusion Constructs to IACs
2.4 Affinity Purification of Biotinylated Proteins
3 Methods
3.1 BirA Fusion Constructs and Controls
3.2 Generation of Stable BirA* Expressing Cell Lines
3.3 Maturation and Cell Surface Expression of BirA* Fusion Constructs
3.4 Localization of BirA* Fusion Constructs and Substrates to IACs
3.5 Proximal Biotinylation and Sample Collection
3.6 Affinity Purification of Biotinylated Proteins
3.7 Identification of Candidate IAC Proteins
4 Notes
References
Chapter 7: Analyzing the Integrin Adhesome by In Situ Proximity Ligation Assay
1 Introduction
2 Materials
2.1 Antibodies
2.2 Duolink In Situ PLA Reagents
2.3 Equipment
2.4 Software for Image Analysis
3 Methods
3.1 Reaction Volumes
3.2 Wash Volumes
3.3 PLA Procedure
3.3.1 Blocking
3.3.2 Primary Antibodies
3.3.3 PLA Probes
3.3.4 Ligation
3.3.5 Amplification
3.3.6 Final Wash Step
3.3.7 Preparation for Imaging
3.3.8 Biological and Technical Controls for In Situ PLA
4 Notes
References
Part III: Biochemical, Proteomics and Computational Methods to Determine Integrin Interactions
Chapter 8: Single-Protein Tracking to Study Protein Interactions During Integrin-Based Migration
Abbreviations
1 Introduction
2 Materials
2.1 Solutions and Reagents
2.2 Other Consumables and Equipment
2.3 Image Acquisition
3 Methods
3.1 Cell Preparation
3.2 Cleaning of Glass Substrates
3.3 Matrix Protein Coatings on Coverslips
3.4 Sample Preparation
3.5 sptPALM Experiments
3.5.1 Single-Protein Tracking Inside and Outside IAS
3.5.2 Single-Protein Tracking Inside and Outside Lamellipodia (See Note 5)
3.5.3 Supercritical Angle Fluorescence and Localization Microscopy
3.5.4 Image Acquisition
3.6 Data Treatment and Analysis
3.6.1 Single-Molecule Localization and Generation of Superresolved Images
3.6.2 Analysis of Single-Protein Tracking Experiments
3.6.3 Fast sptPALM Measurements
3.6.4 sptPALM Acquisition Coupled with Optogenetics
3.6.5 Analysis of SAFe Measurements
3.6.6 Using Mutants of Integrin/Regulators
4 Notes
References
Chapter 9: Biochemical Characterization of the Integrin Interactome
1 Introduction
2 Materials
2.1 Molecular Cloning
2.1.1 Restriction Enzyme Method
2.1.2 Gibson Assembly
2.1.3 Site-Directed Mutagenesis
2.2 Protein Expression
2.2.1 Buffers
2.2.2 General Equipment/Reagents
2.3 Protein Purification
2.3.1 Buffers
Protein Purification for His-Tagged Proteins Using Immobilized Nickel-Affinity Chromatography
Protein Purification by Batch Method for His-Tagged Proteins
Protein Purification by Batch Method for GST-Tagged Proteins
Ion-Exchange Chromatography
2.3.2 General Equipment/Apparatus
2.4 Peptides
2.5 Biochemical Assays
2.5.1 Circular Dichroism (CD)
2.5.2 Nuclear Magnetic Resonance (NMR)
2.5.3 Fluorescence Polarization (FP)
2.5.4 Microscale Thermophoresis (MST)
2.5.5 Size-Exclusion Chromatography-Multiangle Light Scattering (SEC-MALS)
2.5.6 GST-Pulldowns
2.5.7 Actin Cosedimentation Assay
2.5.8 Lipid Cosedimentation Assay
3 Methods
3.1 Molecular Cloning
3.1.1 Restriction Enzyme Digest and Ligation
3.1.2 Gibson Assembly
3.1.3 Site-Directed Mutagenesis
3.2 Protein Expression
3.2.1 Unlabeled Protein Expression
3.2.2 Isotopically Labeled Protein Expression
3.2.3 Condensation Method of Isotopically Labeled Protein Expression
3.3 Protein Purification
3.3.1 Protein Purification for His-Tagged Proteins Using Immobilized Nickel-Affinity Chromatography
3.3.2 Protein Purification for His-Tagged Proteins by Batch Method
3.3.3 Protein Purification by Batch Method for GST-Tagged Proteins
3.3.4 Ion-Exchange Chromatography
3.4 Peptides
3.4.1 Synthetic Peptide Design
3.4.2 Coupling Peptides
3.5 Biochemical Assays
3.5.1 Circular Dichroism (CD)
3.5.2 Measurement of Far-UV CD Spectra
3.5.3 Measurements of Melting Curves
3.5.4 Nuclear Magnetic Resonance (NMR)
3.5.5 Fluorescence Polarization (FP)
3.5.6 Microscale Thermophoresis (MST)
3.5.7 Size-Exclusion Chromatography Multiangle Light Scattering (SEC-MALS)
3.5.8 GST Pulldowns
3.5.9 Actin Cosedimentation Assay
Actin Polymerization
High-Speed Actin-Binding Assay
Low-Speed Actin-Bundling Assay
3.5.10 Lipid Cosedimentation Assays
Preparation of Large Multilamellar Vesicles
Proteins
Interaction Experiment
3.5.11 Structural Studies
4 Notes
References
Chapter 10: Network Analysis of Integrin Adhesion Complexes
1 Introduction
1.1 Integrin Adhesion Complexes
1.2 Network Biology Approaches
2 Materials
2.1 Proteomic Data Processing
2.2 Adhesion Network Analysis
3 Methods
3.1 Proteomic Data Processing
3.1.1 Protein Identification and Quantification
3.1.2 Data Normalization and Missing-Value Imputation
3.2 Adhesion Network Analysis
3.2.1 Active Module Identification
3.2.2 Active Module Weighting
3.2.3 Active Module Clustering
3.2.4 Active Module Visualization
3.2.5 Hub Identification
4 Notes
References
Part IV: Biophysical Methods to Determine Integrin Activation and Its Cellular and Molecular Effects
Chapter 11: Surface Patterning for the Control of Receptor Clustering and Molecular Forces of Integrin-Mediated Adhesions
1 Introduction
2 Materials
2.1 Gold-Micellar Solution
2.2 Polyethylene Glycol Solution
2.3 Peptides and DNA Conjugates
3 Methods
3.1 Glass Coverslip Cleaning
3.2 Spin Coating Procedure of Gold-Polymer Solution on Glass Coverslips
3.3 Hydrogen Plasma Treatment and Analysis of Nanopatterns
3.4 PEGylation of Nanopatterned Coverslips and Click Reaction for the Immobilization of Ligands
3.5 Binding of ITS to Gold Nanoparticles and Covalent Immobilization of Ligands on ITS1
3.6 Fluorescence Microscopy Recording of Molecular Forces
3.7 Data Analysis
4 Notes
References
Chapter 12: Dynamics and Physics of Integrin Activation in Tumor Cells by Nano-Sized Extracellular Ligands and Electromagnetic...
1 Introduction
1.1 Integrins as Mechanosensors
1.2 Thermodynamic and Size Constraints During Integrin Activation
1.3 Quantum Effects in Cells
1.4 Absorption and Emission of EMF Radiation and Energy Storage in Molecular Rotors (MR)
1.5 EMF Energy Storage in Coherent Quantum States in MR
1.6 QED Model of MF Energy Trapped in MR
2 Materials
2.1 Synthesis of NPs
2.2 Tissue Culture
2.3 Determination of NPs Size and Distribution
2.4 Western Blot
3 Methods
3.1 Synthesis of NPs
3.2 Tumor Cell Culture
3.3 Evaluation of the Size Distribution of RE-NPs
3.3.1 X-Ray Diffraction Spectroscopy (XRD) of NPs
3.3.2 Differential Light Scattering (DLS) of NPs
3.3.3 Atomic Force Microscopy (AFM) of NPs
3.3.4 2D-Fast Fourier Transform (FFT) of NPs
3.4 Evaluation of the Formation of Core-Shell NPs by VUV
3.5 Integrin Activation by Western Blotting and Antibodies
3.6 Electric Dipole Interaction Between NPs and LABS
3.6.1 Electric Dipole Polar Force Between NPs and LABS
3.6.2 Electric Dipole Force Radial Component Between NPs and LABS
4 Notes
References
Part V: Integrin Activation and Interactions in Specific Systems and Processes
Chapter 13: Genetic Instruction of Megakaryocytes and Platelets Derived from Human Induced Pluripotent Stem Cells for Studies ...
1 Introduction
2 Materials
2.1 Cell Culture
2.1.1 Culture Reagents and Equipment
2.1.2 Culture Media
2.2 Genetic Modification of imMKCL Cells
2.3 Testing of Genetically Modified Cells
2.3.1 Reagents and Equipment
3 Methods
3.1 Cell Culture
3.1.1 Thawing
3.1.2 Passage
3.1.3 Freezing
3.1.4 Maturation of Human Megakaryocytes and Platelet Production
3.2 Genetic Modification of imMKCL Cells
3.2.1 Nucleofection
3.2.2 Lentiviral Infection
3.3 Testing of Genetically Modified Cells
3.3.1 Validation of imMKCL Maturation and Gene Modification
3.3.2 Flow Cytometry for Determining Megakaryocyte and Platelet Scatter Profiles, and for Analysis of Platelet Surface Adhesio...
3.3.3 Western Blot Analysis of Protein Expression
3.4 Utilization of Genetically Modified Human iPS Cell-Derived Megakaryocytes and Platelets in Integrin-Related Functional Ass...
3.4.1 Flow Cytometric Determination of Fibrinogen Binding to Integrin αIIbβ3
3.4.2 Release of sCD40L from Megakaryocyte and Platelet Alpha Granules
4 Notes
References
Chapter 14: Quantitative Analysis of Integrin Trafficking
1 Introduction
2 Materials
2.1 Buffers and Solutions
2.2 Chemicals and Antibodies
3 Methods
3.1 Cell Surface Integrin Levels
3.2 Internalization Assay
3.3 Recycling Assay
4 Notes
References
Chapter 15: Methods to Study Integrin Functions on Exosomes
1 Introduction
2 Materials
2.1 Exosome Isolation and Characterization
2.1.1 Ultracentrifugation
2.1.2 Density-Gradient Ultracentrifugation
2.1.3 Characterization by NTA
2.2 Cell Culture and Analysis of Exosomal Integrins
2.3 Exosome-Coated Bead Adhesion Assay
2.4 Binding of Free Exosomes to Integrin Ligand Immobilized on Beads
2.5 In Vivo Homing Assay
3 Methods
3.1 Exosome Isolation and Characterization
3.1.1 Ultracentrifugation
3.1.2 Density-Gradient Ultracentrifugation
3.1.3 Characterization by Nanoparticle Tracking Analysis (NTA)
3.2 Cellular Assays Using Exosomes
3.2.1 Immobilization of Exosomes to Beads
3.2.2 Measurement of Exosomal Integrin Levels Using Immunofluorescent Flow-Cytometry
3.3 V-Well Adhesion Assay for Exosome-Coated Beads
3.3.1 Immobilization of Fibronectin or Laminin on a V-Well Microtiter Plate
3.3.2 Measurement of Exosome Adhesion
3.4 Binding of Exosomes to Integrin Ligand Immobilized on Beads
3.5 In Vivo Competitive Exosome Homing Assay
4 Notes
References
Part VI: Integrin Ligands and the Extracellular Matrix
Chapter 16: Functional Bioinformatics Analyses of the Matrisome and Integrin Adhesome
1 Introduction
2 Materials
3 Methods
3.1 Data Preprocessing
3.2 Comparison to In Silico Descriptions of the ECM and Adhesome
3.3 Interaction Networks
3.4 Generating Functional Enrichment Maps
3.5 Functional Analyses Using Gene Ontology
4 Notes
References
Chapter 17: Quantifying Polarized Extracellular Matrix Secretion in Cultured Endothelial Cells
1 Introduction
2 Materials
3 Methods
3.1 siRNA-mediated Gene Silencing in AECs
3.2 Preparing FN-free EGM-2 Medium
3.3 Seeding AECs on Transwell Chambers
3.4 Check Integrity of Endothelial Cell Monolayer
3.5 Fibronectin Secretion Quantification
3.6 Immunofluorescence on Transwell Membranes
4 Notes
References
Index