Elsevier S & T

Integrins

Cheresh, David A.

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Table of contents
  • Contentsv
  • Contributorsxiii
  • Prefacexix
  • Volumes in Seriesxxi
  • Chapter 1: Quantitative Measurements of Integrin-Mediated Adhesion to Extracellular Matrix1
  • 1. Introduction2
  • 2. Spinning Disc Measurements3
  • 3. Protocols4
  • 3.1. Preparation of the substrate4
  • 3.2. Preparation of the cells7
  • 3.3. Setup of the spinning disc device7
  • 3.4. Counting the discs9
  • 3.5. Generating adhesion plots using SigmaPlot10
  • 4. Data Analysis12
  • 4.1. Interpretation and sources of error12
  • 4.2. Quantification of other parameters14
  • 4.3. Equilibrium and rates16
  • 4.4. Making comparisons18
  • Acknowledgments19
  • Appendix 1. Reagents19
  • Appendix 2. Spinning Disc Device20
  • References24
  • Chapter 2: Investigating Integrin Regulation and Signaling Events in Three-Dimensional Systems27
  • 1. Introduction28
  • 1.1. Types of 3D matrices29
  • 1.2. Challenges of working in 3D matrices30
  • 2. Collagen Matrices of Different Densities31
  • 3. Gel Contraction as a Measure to Quantify Cell Contractility33
  • 4. Rho Activity Assay from Cells Cultured in 3D Collagen Gels33
  • 4.1. Cell culture34
  • 4.2. G-LISA assay34
  • 5. Co-Immunoprecipitation of Integrin-Associated Proteins from Cells Cultured in 3D Collagen Gels35
  • 6. beta1-Integrin Endocytosis Assays36
  • 6.1. Time-lapse imaging of integrin trafficking in cells cultured on 2D surfaces36
  • 6.2. Endosome protection biotinylation assay39
  • 7. Imaging Cell-Matrix Interactions in 3D Collagen Gels39
  • 7.1. Cell culture40
  • 7.2. Stable transfection40
  • 7.3. Transient transfection40
  • 7.4. Three-dimensional collagen gels43
  • 7.5. Nonlinear optical imaging of collagen-3D matrix adhesion interaction43
  • References197
  • Chapter 3: Integrins in Cell Migration47
  • 1. Introduction47
  • 2. Methods for Analysis of Integrin-Mediated Cell Migration49
  • 2.1. Imaging of adhesion dynamics50
  • 2.2. Imaging of podosomes57
  • References197
  • Chapter 4: Integrin Cytoskeletal Interactions69
  • 1. Introduction70
  • 2. Models of Integrin Cytoplasmic Tails72
  • 3. Expression Constructs for Integrin Tail Model Proteins74
  • 4. Purification of Integrin Cytoplasmic Tails74
  • 5. Preparation of the Affinity Matrix76
  • 6. Binding Assays Using Cell Lysates77
  • 6.1. Cell lysis77
  • 6.2. Binding assays77
  • 7. Direct Protein-Protein Binding Assays78
  • 7.1. Production of GST-fusion proteins78
  • 7.2. Binding assays with purified GST-fusion proteins79
  • 8. Advanced Applications79
  • 8.1. Competition assays79
  • 8.2. Investigation of the role of integrin phosphorylation80
  • 8.3. ELISA using model tail proteins80
  • 8.4. Surface plasmon resonance assays80
  • 8.5. Structural studies of model proteins81
  • 9. Concluding Remarks81
  • Acknowledgments81
  • References197
  • Chapter 5: Cell Survival in a Three-Dimensional Matrix85
  • 1. Introduction86
  • 2. Protocols87
  • 2.1. Isolation of matched cell lines expressing differing levels of integrins87
  • 2.2. Preparation of three-dimensional collagen culture89
  • 2.3. Scoring apoptosis in collagen gels91
  • 2.4. Immunofluorescence techniques for assessing cell survival93
  • 2.5. Determining cell survival by caspase activation94
  • 2.6. Adaptation of three-dimensional culture to existing immunodetection methods95
  • 2.7. Preparation of three-dimensional fibrin cell culture96
  • 2.8. Alternative approaches and concerns97
  • 2.9. Studies on cells cultured on the surface of three-dimensional gels98
  • 3. Conclusions and Perspective100
  • References100
  • Chapter 6: Platelet Integrin Adhesive Functions and Signaling103
  • 1. Introduction104
  • 2. Cell Models104
  • 2.1. Platelets104
  • 2.2. Cell lines106
  • 3. Microscopy107
  • 3.1. Cell spreading assay107
  • 3.2. Immunostaining107
  • 4. Assaying Integrin Adhesive Responses108
  • 4.1. Flow cytometry analysis of integrin function108
  • 4.2. Adhesion assay110
  • 4.3. Platelet aggregation110
  • 4.4. Clot retraction111
  • 5. Biochemical Analysis of Integrin-Based Signaling111
  • 5.1. Initiation of integrin alphaIIbbeta3 "outside-in" signaling111
  • 5.2. Immunoprecipitation and co-immunoprecipitation112
  • References114
  • Chapter 7: Development of Monoclonal Antibodies to Integrin Receptors117
  • 1. Introduction118
  • 1.1. Monoclonal antibodies to integrin receptors119
  • 1.2. Production of monoclonal antibodies with hybridoma technology120
  • 2. Methods121
  • 2.1. Production of monoclonal antibodies to integrin receptors using hybridoma technology121
  • 2.2. Choice of the antigen (native versus non-native receptor)122
  • 2.3. Choice of a test system127
  • 2.4. Immunization strategy129
  • 2.5. Choice of adjuvant and immunization protocol130
  • 2.6. Polyclonal antisera screening133
  • 2.7. Preparation of the spleen for fusion135
  • 2.8. Design the strategy for screening the fusion135
  • 2.9. Screen fusions via a cell-adhesion assay136
  • 2.10. Biochemical characterization of anti-integrin antibodies by immune precipitation137
  • 2.11. Identification of integrin receptors by immune precipitation/Western blot139
  • 3. Protocols139
  • 3.1. Cell-based ELISA139
  • 3.2. Cell adhesion assay140
  • 3.3. Production of monoclonal antibodies via cell fusion143
  • 3.4. Cloning146
  • 3.5. Isotyping146
  • 3.6. Immunoprecipitation for integrin identification147
  • 3.7. Preclearing of lysates148
  • 3.8. Preparation and storage of protein A agarose148
  • 3.9. Coupling of rabbit anti-mouse IgG to protein A-agarose148
  • 3.10. Coupling of test mouse antibody-containing supernatants to rabbit anti-mouse IgG protein A-aga149
  • 3.11. Immunoprecipitation of cell lysate149
  • 3.12. Releasing immunoprecipitated material from beads150
  • 3.13. Western blot to detect biotinylated and immune-precipitated integrin receptors150
  • 3.14. IP buffers151
  • References151
  • Chapter 8: Cell Adhesion, Cellular Tension, and Cell Cycle Control155
  • 1. Introduction156
  • 2. Preparative Methods156
  • 2.1. Cell culture156
  • 2.2. Transfection, adenoviral infection, and siRNA158
  • 2.3. Preparing substrata of near physiological compliance using matrix proteins linked to polyacryla162
  • 3. Analytical Methods165
  • 3.1. Cell cycle analysis by propidium iodide staining and flow cytometry165
  • 3.2. Crystal violet staining to monitor cell proliferation165
  • 3.3. Immunofluorescence166
  • 3.4. Rho family GTPase pulldown assays168
  • 3.5. Assays for transcriptional regulation169
  • 3.6. Special analytical procedures needed for experimentation on acrylamide-extracellular matrix sub174
  • Acknowledgments174
  • References174
  • Chapter 9: Analysis of Integrin Signaling by Fluorescence Resonance Energy Transfer177
  • 1. Introduction178
  • 2. Fluorescence Proteins and FRET179
  • 2.1. Fluorescence proteins179
  • 2.2. RET179
  • 3. Integrin Signaling180
  • 3.1. Integrin activation180
  • 3.2. Integrin-related signaling cascades181
  • 4. FRET Analysis of Integrin Signaling182
  • 4.1. FRET visualization of integrin activation182
  • 4.2. FRET visualization of the activation of integrin-related signaling molecules182
  • 5. Future Directions196
  • Acknowledgments197
  • References197
  • Chapter 10: Studies on Integrins in the Nervous System203
  • 1. Introduction204
  • 2. Neuronal Cell Adhesion and Neurite Outgrowth Assays208
  • 2.1. Preparation of substrates208
  • 2.2. Cell adhesion assays209
  • 2.3. Neurite outgrowth assays209
  • 2.4. Axons versus dendrite quantification210
  • 3. Neuronal Culture Procedures210
  • 3.1. Rat PC12 pheochromocytoma cell culture210
  • 3.2. Rodent sympathetic neuron cultures211
  • 3.3. Rodent DRG sensory neuron cultures211
  • 3.4. Rodent trigeminal sensory neuron cultures212
  • 3.5. Rodent hippocampal neuron cultures212
  • 3.6. Rodent cortical neuron cultures213
  • 3.7. Chick DRG sensory neuron culture213
  • 3.8. Chick ciliary neuron culture213
  • 3.9. Chick motor neuron culture213
  • 3.10. Chick retinal neuron culture214
  • 4. Biochemical Studies Using Cultured Neurons214
  • 4.1. Surface labeling of integrins and other neuronal glycoproteins (used for DRG and ciliary gangli215
  • 4.2. Immunocytochemistry216
  • References
  • Chapter 11: Methods for Identifying Novel Integrin Ligands223
  • 1. Introduction224
  • 2. Production of Soluble alpha8tbeta1-AP228
  • 2.1. Construction of secreted integrin expression vectors228
  • 2.2. Purification of soluble truncated integrin heterodimers229
  • 3. Solid-Phase Binding Assays with Soluble Integrin Heterodimers230
  • 4. Histochemistry with Soluble Integrin Heterodimers231
  • 5. Far Western Blotting Using Integrin Heterodimers231
  • 6. Ligand Detection Using Intact Integrin Receptors232
  • 7. alpha3beta 1 Immunolabeling and Purification233
  • 8. Receptor-Binding Assays234
  • References234
  • Chapter 12: Analysis of Integrin Functions in Peri-Implantation Embryos, Hematopoietic System, and S239
  • 1. Introduction240
  • 2. Analysis of Integrin Functions during Peri-Implantation Development241
  • 2.1. Generation of EBs from ESC aggregates243
  • 2.2. Immunocytochemical characterization of EBs
  • 3. Analysis of Integrin Functions in Blood249
  • 3.1. Generation of bone marrow chimeras250
  • 3.2. Standard flow cytometric analysis of cell surface receptors252
  • 3.3. Flow cytometric lacZ staining of hematopoietic cells254
  • 3.4. Generation of dendritic cells from bone marrow256
  • 3.5. In vitro T-cell proliferation assay258
  • 3.6. In vivo T-cell proliferation assay261
  • 3.7. Induction of experimental autoimmune encephalomyelitis in C57/BL6 mice262
  • 3.8. Mononuclear cell isolation from the central nervous system263
  • 4. Analysis of Integrin Functions in Skin264
  • 4.1. Isolation of mouse skin267
  • 4.2. Embedding of skin in cryomatrix and preparation of cryosections269
  • 4.3. Immunofluorescence staining of skin cryosections270
  • 4.4. LacZ staining on cryosections273
  • 4.5. Embedding and cutting of skin for paraffin sections274
  • 4.6. H&E staining on paraffin sections275
  • 4.7. Evaluation of keratinocyte proliferation by BrdU incorporation method276
  • 5. In Vitro Skin Analysis277
  • 5.1. Isolation of primary keratinocytes277
  • 5.2. Detachment of keratinocytes from cell culture dishes279
  • 5.3. Analysis of expression and activation of integrins on freshly isolated or cultured keratinocyte280
  • 5.4. Analysis of fibronectin fragment binding by primary keratinocytes280
  • 5.5. Assessment of integrin-dependent adhesion to ECM proteins282
  • 5.6. Analysis of integrin-mediated spreading of primary keratinocytes283
  • 5.7. In vitro wound-healing assay with primary keratinocytes283
  • 5.8. Trans-well migration assay with primary keratinocytes284
  • 5.9. Immunfluorescence on primary keratinocytes285
  • References
  • Chapter 13: Identification and Molecular Characterization of Multiple Phenotypes in Integrin Knockou291
  • 1. Introduction292
  • 2. Strategies for Generating Integrin Knockout Mice292
  • 3. Validation of Observed Phenotypes by Transgenic Rescue In Vivo293
  • 4. In Vitro Transgenic Rescue294
  • 5. Generation of Blocking Mouse Monoclonal Antibodies that Recognize Murine Integrins294
  • 6. Selection of Integrin Knockout Lines to Study295
  • 7. Selection of Phenotypic Assays296
  • 8. Identification of Additional Phenotypes in Integrin Knockouts that Survive Embryonic Development297
  • 8.1. Pattern of distribution298
  • 9. Identification of Integrin Knockout Phenotypes Based on Educated Guesses from in Vitro Experiment299
  • 10. Use of Expression Microarrays to Suggest Possible Phenotypic Assays300
  • 11. Summary302
  • References
  • Chapter 14: Purification, Analysis, and Crystal Structure of Integrins307
  • 1. Introduction308
  • 2. Purification and Analysis of Integrin Heterodimers308
  • 2.1. Integrin isolation309
  • 2.2. Integrin characterization315
  • 3. Atomic Structure of Integrins Using Macromolecular Crystallography320
  • 3.1. Crystal structure determination of the integrin ectodomain320
  • 3.2. Crystal structure determination of alphaA domain327
  • 4. Summary328
  • Acknowledgments
  • References
  • Chapter 15: Electron Microscopy of Integrins337
  • 1. Introduction338
  • 2. Purification of Full-Length alphaIIbbeta3 from Platelets339
  • 3. Preparation of Specimens for Negative-Stain EM341
  • 4. Preparation of Specimens for Cryo-EM346
  • 5. Low-Dose EM348
  • 6. Image Appraisal, Particle Selection, and Preprocessing350
  • 7. Initial Particle Analysis and Generation of Reference-Free Aligned Class Averages355
  • 8. Three-Dimensional Structure Refinement357
  • 9. Evaluation of the Refinement362
  • 10. Pseudoatomic Modeling of EM Density Maps365
  • 11. Future Prospects for Cryo-EM369
  • Acknowledgments
  • References
  • Chapter 16: Intravital Imaging and Cell Invasion375
  • 1. Introduction376
  • 1.1. Overview376
  • 1.2. Background and applications378
  • 2. Methods380
  • 2.1. Basic configuration and fabrication of the window chamber380
  • 2.2. Preparation of the window chamber hardware381
  • 2.3. Specific variations in the window chamber for tumor studies382
  • 2.4. Surgical installation of the dorsal skinfold window chamber383
  • 2.5. Cell lines and preparation of cells for placement into window chamber tissue390
  • 2.6. Imaging of the window chambers391
  • Acknowledgments
  • References
  • Chapter 17: Using Xenopus Embryos to Investigate Integrin Function403
  • 1. Introduction404
  • 2. Visualization of Integrins, Extracellular Matrix, and Cytoskeleton in Embryo Explants405
  • 2.1. Embryo fixation and microsurgery405
  • 2.2. Methods for immunostaining of Xenopus explants for confocal microscopy407
  • 2.3. Visualization of actin in Xenopus explants407
  • 2.4. Microscopy: general considerations409
  • 3. Live Imaging of Fibronectin Fibrils410
  • 3.1. Preparation of thin agarose sheets412
  • 3.2. Explant preparation412
  • 3.3. Live imaging of FN fibrils413
  • 4. Conclusions413
  • Acknowledgments
  • References
  • Chapter 18: Methods to Study Lymphatic Vessel Integrins415
  • 1. Introduction416
  • 2. Isolation of Human Lymphatic Endothelial Cells418
  • 3. Isolation of Murine Lymphatic Endothelial Cells418
  • 4. Characterization of LEC Integrin Expression421
  • 5. In Vitro Cell Adhesion Assays421
  • 6. Migration Assays423
  • 7. Matrigel Tube Formation424
  • 8. Frozen Section Immunofluorescence Microscopy425
  • 9. Paraffin-Embedded Section Immunohistochemistry427
  • 10. Growth-Factor-Induced Lymph Node Lymphangiogenesis428
  • 11. Murine Lymphangioma Model428
  • 12. Tumor Models430
  • 13. Endothelial Cell-Specific Integrin alpha4 Deletion Mutant432
  • 14. Intravital Microscopy of Lymph Nodes433
  • 15. Abdominal Window Implantation433
  • 15.1. Imaging inguinal window chamber mice434
  • 15.2. Imaging of vascular and lymphatic vessel perfusion434
  • 16. Intradermal and Footpad Injections436
  • References436
  • Chapter 19: Analysis of Integrin Signaling in Genetically Engineered Mouse Models of Mammary Tumor P439
  • 1. Introduction440
  • 2. Experimental Approach442
  • 3. Measurement of Primary Tumor Growth443
  • 4. Spontaneous and Allograft Lung Metastasis Assays444
  • 5. Analysis of Tumor Sections448
  • 5.1. Methods450
  • 6. Detergent Extraction of Tumor Samples452
  • 7. Ex Vivo Culture of Mammary Tumor Cells452
  • 7.1. Method453
  • 8. Genetic Modification of Primary Mammary Tumor Cells453
  • 8.1. Production of retroviral/lentiviral stocks and cell infection in vitro455
  • 9. Disruption of Epithelial Adhesion and Growth Control456
  • 9.1. Cell proliferation assay456
  • 9.2. Apoptosis assay456
  • 9.3. Matrigel invasion assay457
  • 9.4. Culture in three-dimensional matrigel457
  • Acknowledgments
  • References459
  • Chapter 20: Design and Chemical Synthesis of Integrin Ligands463
  • 1. Introduction464
  • 2. Design of Integrin Ligands: Overview465
  • 2.1. Ligand-based design466
  • 2.2. Rational structure-based design475
  • 3. Application of Integrin Ligands for Imaging and Surface Coating480
  • 3.1. Coating of material surfaces480
  • 3.2. Tumor imaging and therapy484
  • 4. Experimental Section488
  • 4.1. General procedures488
  • 4.2. Preparation of cyclo(-RGDfNMeVal)-)492
  • 4.3. Synthesis of aza-glycine mimetic 2492
  • References495
  • Chapter 21: Evaluating Integrin Function in Models of Angiogenesis and Vascular Permeability505
  • 1. Introduction506
  • 1.1. Selective expression of alphavbeta3 on angiogenic blood vessels507
  • 1.2. Distinct functions for alphavbeta3 and alphavbeta5 during angiogenesis and vascular leak508
  • 1.3. Role of beta1 integrins in angiogenesis and vascular permeability509
  • 1.4. Role of a6b4 in angiogenesis510
  • 1.5. Endogenous negative regulators of integrin function510
  • 1.6. Clinical testing of integrin antagonists as antiangiogenic agents511
  • 2. Assessing Role of Integrins During Angiogenesis511
  • 2.1. In vitro models of angiogenesis512
  • 2.2. Chick chorioallantoic membrane angiogenesis assay513
  • 2.3. Matrigel plug angiogenesis assay in the mouse513
  • 2.4. Assessing angiogenesis using intravital microscopy515
  • 2.5. Ocular angiogenesis models515
  • 2.6. Evaluating angiogenesis in the zebrafish517
  • 3. Assessing Role of Integrins in Vascular Permeability518
  • 3.1. In vitro models to assess vascular permeability518
  • 3.2. The "Miles assay" to assess leak in the skin519
  • 3.3. Evaluation of biochemical signals during the vascular leak response520
  • 3.4. Evaluation of structural and ultrastructural changes during the vascular leak response520
  • 4. Concluding Remarks521
  • 4.1. Studying angiogenesis and vascular leak in models of cancer521
  • 4.2. Acknowledging and understanding diversity among subjects522
  • 4.3. Necessity for better tools to study integrin function523
  • References523
  • Author Index529
  • Subject Index559
Book details
  • Vendor Elsevier S & T
  • SKU 9780123739247
  • ISBN-13 9780080551388
  • Author Cheresh, David A.
  • Category Medical
  • Subject Biochemistry

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An integrin, or integrin receptor, is an integral membrane protein in the plasma membrane of cells. It plays a role in the attachment of a cell to the extracellular matrix (ECM) and to other cells, and in signal transduction from the ECM to the cell. There are many types of integrin, and many cells have multiple types on their surface. Integrins are of vital importance to all metazoans, from humans to sponges. This volume in Methods in Enzymology presents methods for studying integrins.