Intermediate Filament Cytoskeleton

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ISBN: 9780125641739 | 0125641737
Cover: Hardcover
Copyright: 12/2/2004

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Intermediate filaments are a large family of proteins that are the cytoskeletal elements involved in a number of skin, liver, neuromuscular, cardiac, eye and hair diseases. Intermediate filament genes are regulated in a tissue-and cell type-specific manner and their polymerized protein products protects the cells and tissue they are part of against a variety of mechanical and nonmechanical stresses. This book provides a comprehensive resource of methodology essentials, describing a variety of essential tools and assays for studying intermediate filaments. The book provides user-friendly advice and protocols covering all aspects of intermediate filaments including protein isolation and structure, protein and gene regulation, relationship to disease and apoptosis, and associated proteins. Both mammalian and non-mammalian systems and animal models are covered, making this book a must-have for any investigator wishing to study IF genes or their protein products.

Contributors	p. xv
Preface	p. xxi
Dedication	p. xxvii
Acknowledgment	p. xxix
General Methods to Study Intermediate Filament Proteins &3- From Molecules to Disease
Isolation, Characterization, and In Vitro Assembly of Intermediate Filaments
Introduction	p. 3
Materials and Methods	p. 6
Pearls and Pitfalls	p. 19
Concluding Remarks	p. 20
References	p. 21
Intermediate Filament Protein Structure Determination
Introduction	p. 25
Approaches and Methods	p. 26
Pearls and Pitfalls	p. 39
Concluding Remarks	p. 40
References	p. 40
Intracellular Microrheology as a Tool for the Measurement of the Local Mechanical Properties of Live Cells
Introduction	p. 46
Materials and Instrumentation	p. 53
Procedures	p. 54
Pearls and Pitfalls	p. 60
Concluding Remarks	p. 61
References	p. 62
Developing Mouse Models to Study Intermediate Filament Function
Introduction	p. 65
Essential Materials and Methods	p. 67
Websites, Tables, Reagents, and Vendors of Interest	p. 91
Reference	p. 92
Uncovering the Roles of Intermediate Filaments in Apoptosis
Introduction	p. 96
Materials and Instrumentation	p. 106
Procedures	p. 109
Pearls and Pitfalls	p. 120
Conclusion and Perspectives	p. 121
References	p. 122
Molecular Genetics Methods for Human Intermediate Filament Diseases
Introduction	p. 132
Materials and Instrumentation	p. 139
Procedures	p. 139
Pearls and Pitfalls	p. 152
Discussion and Concluding Remarks	p. 154
References	p. 158
Intermediate Filaments and Multiparameter Flow Cytometry for the Study of Solid Tumors
Introduction	p. 163
Materials and Instrumentation	p. 177
Procedures	p. 191
Comments	p. 196
Pearls and Pitfalls	p. 198
References	p. 199
Intermediate Filament Protein Inclusions
Introduction	p. 206
Materials and Instrumentation	p. 216
Procedures	p. 218
Pitfalls	p. 224
Discussion	p. 224
References	p. 225
Stress Models for the Study of Intermediate Filament Function
Introduction	p. 230
Materials and Instrumentation	p. 249
Procedures	p. 251
Pitfalls	p. 256
Discussion/Concluding Remarks	p. 258
References	p. 258
General Methods to Study Intermediate Filament Proteins and Gene Regulation
Regulation of Intermediate Filament Gene Expression
Introduction	p. 267
Materials	p. 277
Procedures	p. 278
Pitfalls	p. 292
Discussion and Concluding Remarks	p. 293
References	p. 293
Fluorescence-Based Methods for Studying Intermediate Filaments
Introduction	p. 298
Materials and Methods	p. 301
Pearls and Pitfalls	p. 310
Discussion	p. 313
References	p. 318
Imaging of Keratin Dynamics during the Cell Cycle and in Response to Phosphatase Inhibition
Introduction	p. 322
Materials and Instrumentation	p. 324
Procedures	p. 327
Pearls and Pitfalls	p. 344
Discussion and Concluding Remarks	p. 348
References	p. 349
Approaches to Study Phosphorylation of Intermediate Filament Proteins Using Site-Specific and Phosphorylation State-Specific Antibodies
Introduction	p. 354
Materials and Instrumentation	p. 355
Procedures	p. 357
Pearls and Pitfalls	p. 369
Discussion and Concluding Remarks	p. 370
References	p. 370
Approaches to Study Posttranslational Regulation of Intermediate Filament Proteins
Introduction	p. 374
Materials and Methods: Characterization of Intermediate Filament Phosphorylation	p. 391
Pearls and Pitfalls	p. 403
Conclusions	p. 404
References	p. 404
Methods to Study Specific Intermediate Filament Proteins in Mammalian Systems
Hair Keratins and Hair Follicle-Specific Epithelial Keratins
Introduction	p. 414
Procedures and Comments	p. 417
Pitfalls and Problems	p. 445
References	p. 448
Skin: An Ideal Model System to Study Keratin Genes and Proteins
Introduction	p. 454
Collection of Mouse Skin Tissue for Analysis	p. 462
Preparation of Tissues for Morphological Studies	p. 463
Cell Culture Studies	p. 468
Preparation and Analysis of Protein and RNA from Tissues and Cells	p. 473
Pearls and Pitfalls	p. 477
Conclusion	p. 480
References	p. 484
Studying Simple Epithelial Keratins in Cells and Tissues
Introduction	p. 489
Materials and Methods	p. 492
Pearls and Pitfalls	p. 511
Concluding Remarks	p. 513
References	p. 514
Muscle Intermediate Filament Proteins
Introduction	p. 519
Materials	p. 524
Procedures	p. 525
Pearls and Pitfalls	p. 547
Concluding Remarks	p. 548
References	p. 549
[superscript 35 S]Methionine Metabolic Labeling to Study Axonal Transport of Neuronal Intermediate Filament Proteins In Vivo
Introduction	p. 555
Materials and Instrumentation	p. 560
Procedures	p. 561
Comments	p. 565
Pitfalls	p. 566
References	p. 566
Lamins
Introduction	p. 574
Materials	p. 576
Purification of Lamins Produced in Bacteria	p. 579
Production of Lamins in Insect Cells Using the Baculovirus System	p. 586
Selection of Cultured Cells and Tissues for Lamin Enrichment	p. 588
Enrichment of Lamins from Isolated Mammalian Liver Nuclei	p. 588
Enrichment of Lamins from Cultured Cells (Tested for Xenopus A6 Cells and HeLa Cells)	p. 591
Immunoprecipitation of Lamin from Cell Extracts	p. 593
References	p. 594
The Intermediate Filament Systems in the Eye Lens
Introduction	p. 598
Materials and Instrumentation	p. 603
Procedures	p. 607
Pearls and Pitfalls	p. 620
Discussion and Concluding Remarks	p. 620
References	p. 621
Methods to Study Specific Intermediate Filament Proteins in Non-Mammalian Systems
Fish Keratins
Introduction	p. 627
Materials, Procedures, and Pitfalls	p. 630
Discussion and Concluding Remarks	p. 663
References	p. 668
Performing Functional Studies of Xenopus laevis Intermediate Filament Proteins Through Injection of Macromolecules into Early Embryos
Introduction	p. 674
Instrumentation and Materials	p. 679
Procedures	p. 686
Pearls and Pitfalls	p. 689
Discussion and Concluding Remarks	p. 691
References	p. 695
Intermediate Filaments in Caenorhabditis elegans
Introduction	p. 704
Intermediate Filaments in C. elegans	p. 704
General Methodology	p. 707
Forming Intermediate Filaments In Vitro	p. 708
Indirect Immunofluorescence Analysis	p. 709
Immunogold-Electron Microscopy Preembedding Staining of C. elegans Embryos	p. 711
Sample Preparation for Transmission Electron Microscopy Analysis of C. elegans Embryos	p. 713
Live Imaging of C. elegans	p. 714
References	p. 716
Methods to Study Intermediate Filament Associated Proteins
Plectin
Introduction	p. 722
Materials and Methods	p. 731
Pearls and Pitfalls	p. 744
Concluding Remarks	p. 750
References	p. 750
In Vitro Methods for Investigating Desmoplakin-Intermediate Filament Interactions and Their Role in Adhesive Strength
Introduction	p. 758
Materials and Methods	p. 759
Pitfalls and Pearls	p. 780
Discussion	p. 783
References	p. 784
Studying Cytolinker Proteins
Introduction	p. 787
Methods for Studying Cytolinker Proteins	p. 789
Pearls and Pitfalls	p. 811
Concluding Remarks	p. 814
References	p. 815
Keratin Bundling Proteins
Introduction	p. 817
Methods	p. 822
Pearls and Pitfalls	p. 825
Concluding Remarks	p. 825
References	p. 826
Lamin-Associated Proteins
Introduction	p. 829
Materials	p. 847
Procedures	p. 847
Pearls and Pitfalls	p. 853
Discussion and Concluding Remarks	p. 854
References	p. 854
Index	p. 861
Volumes in Series	p. 879
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