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Intermediate Filament Associated Proteins

1st Edition - January 14, 2016

Editors: Katherine L. Wilson, Arnoud Sonnenberg

Hardback ISBN:

9 7 8 - 0 - 1 2 - 8 0 3 4 6 9 - 9

eBook ISBN:

9 7 8 - 0 - 1 2 - 8 0 3 4 9 0 - 3

Intermediate Filament Associated Proteins, the latest volume in the Methods in Enzymology series, continues the legacy of this premier serial with quality chapters authored by… Read more

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Intermediate Filament Associated Proteins, the latest volume in the Methods in Enzymology series, continues the legacy of this premier serial with quality chapters authored by leaders in the field. This volume covers research methods in intermediate filament associated proteins and contains sections on such topics as lamin-associated proteins, intermediate filament-associated proteins and plakin, and other cytoskeletal cross-linkers.

Preface
Part I: Identification and Biochemical Analysis of Lamin-Associated Proteins
- Chapter One: BioID Identification of Lamin-Associated Proteins
  - Abstract
  - 1 Introduction
  - 2 BioID Method
  - 3 Anticipated Results
  - 4 Conclusions
  - Acknowledgment
- Chapter Two: Proximity-Dependent Biotin Identification (BioID) in Dictyostelium Amoebae
  - Abstract
  - 1 Introduction
  - 2 Materials
  - 3 Methods
  - 4 Conclusions
- Chapter Three: Purification and Structural Analysis of LEM-Domain Proteins
  - Abstract
  - 1 Introduction
  - 2 “Divide and Conquer” Approach to Study LEM-Domain Proteins
  - 3 Analysis of Predicted Unstructured Regions in LEM-Domain Proteins
  - 4 LEM-Domain Proteins with Their Partners
  - 5 Concluding Remarks
  - Acknowledgments
- Chapter Four: Purification and Structural Analysis of SUN and KASH Domain Proteins
  - Abstract
  - 1 Introduction
  - 2 Purification of SUN Proteins and SUN–KASH Complexes
  - 3 Structural Analysis of Human SUN2 and SUN2–KASH1/2 Complexes
  - 4 Conclusions and Pitfalls
  - Acknowledgments
- Chapter Five: Purification of Lamins and Soluble Fragments of NETs
  - Abstract
  - 1 Introduction
  - 2 Purification Parameters
  - 3 Lamin A Purification
  - 4 NET Fragment Purification
  - 5 Gel Extraction of Proteins
  - 6 Conclusions and Potential Applications
  - Acknowledgments
- Chapter Six: Simple Separation of Functionally Distinct Populations of Lamin-Binding Proteins
  - Abstract
  - 1 Introduction
  - 2 Cell Culture (with or without Transfection) and Harvesting
  - 3 Separate Nuclei from Cytoplasm
  - 4 Fractionate Nuclei to Separate “Easy-” Versus “Sonication-Dependent” Proteins
  - 5 Analysis by Immunoprecipitation and Immunoblotting
  - 6 Conclusions
  - Acknowledgments
Part II: General Methods for Analysis of Intermediate Filament-Associated Proteins
- Chapter Seven: Development of a Novel Green Fluorescent Protein-Based Binding Assay to Study the Association of Plakins with Intermediate Filament Proteins
  - Abstract
  - 1 Introduction
  - 2 Recombinant Proteins
  - 3 Fluorescence Measurement of Soluble EGFP
  - 4 Expression of EGFP-Fusion Proteins in Mammalian Cells
  - 5 Analysis of the Interaction of EGFP-Fusion Recombinant Plakins with IF Proteins
  - 6 Concluding Remarks
  - Acknowledgments
- Chapter Eight: Functional Analysis of Keratin-Associated Proteins in Intestinal Epithelia: Heat-Shock Protein Chaperoning and Kinase Rescue
  - Abstract
  - 1 Introduction
  - 2 Isolation of Keratin Intermediate Filaments for Functional Assays
  - 3 Detection of Chaperoning Activity on Keratin Intermediate Filaments, Relative to Cytosolic Activity
  - 4 Refolding of aPKC by Hsp70/40 in Keratin Intermediate Filaments
  - 5 Supplementation Assays Using Cytosol (S) and Isolated Keratin Intermediate Filaments (kP)
  - 6 Summary
  - Acknowledgments
- Chapter Nine: Purification of Protein Chaperones and Their Functional Assays with Intermediate Filaments
  - Abstract
  - 1 Introduction
  - 2 Purification of sHSPs
  - 3 Purification of IF Proteins
  - 4 Analysis of sHSP and IF Proteins Interaction
  - Acknowledgments
- Chapter Ten: Purification and Structural Analysis of Plectin and BPAG1e
  - Abstract
  - 1 Introduction
  - 2 Purification of Fragments of Plectin and BPAG1e for Structural Analysis
  - 3 Structural Analysis of Plectin and BPAG1e
  - 4 Analysis of Multi-Domain Structures Combining MX, DEER, and SAXS
  - 5 Conclusion
  - Acknowledgments
- Chapter Eleven: Purification and Structural Analysis of Desmoplakin
  - Abstract
  - 1 Introduction
  - 2 Purification and Structural Analysis of DPNT
  - 3 Purification and Structural Analysis of DPCT
  - 4 Future Directions
  - Acknowledgments
- Chapter Twelve: Degradation of the Intermediate Filament Family by Gigaxonin
  - Abstract
  - 1 Introduction
  - 2 Gigaxonin Expression and Localization
  - 3 Analyzing Gigaxonin Role in IF Organization
  - 4 Future Perspectives
  - Acknowledgments
Part III: Functional and Genetic Analysis of the Plakin and Other Cytoskeletal Cross-Linkers
- Chapter Thirteen: Functional and Genetic Analysis of Plectin in Skin and Muscle
  - Abstract
  - 1 Introduction
  - 2 Main Challenges
  - 3 Tools
  - 4 Methods, Pearls, and Pitfalls
  - 5 Conclusions
  - Acknowledgments
- Chapter Fourteen: Functional and Genetic Analysis of Epiplakin in Epithelial Cells
  - Abstract
  - 1 Introduction
  - 2 Main Considerations
  - 3 Methods: Pearls and Pitfalls
  - 4 Conclusions
  - Acknowledgments
- Chapter Fifteen: In Vitro Model of the Epidermis: Connecting Protein Function to 3D Structure
  - Abstract
  - 1 Introduction
  - 2 Historical Perspective
  - 3 Cellular Constituents of 3D Raft Cultures
  - 4 3D Raft Cultures
  - 5 Experimental Manipulation of the Organotypic Raft Cultures
  - 6 Analysis of Organotypic Raft Cultures and Other Methods
  - 7 Conclusions
  - Acknowledgments
- Chapter Sixteen: Functional Analysis of Periplakin and Envoplakin, Cytoskeletal Linkers, and Cornified Envelope Precursor Proteins
  - Abstract
  - 1 Introduction
  - 2 Implications of Plakin Domain Structure to Functional Analysis
  - 3 Analysis of the Plakin Expression and Localization in Cell Culture Models
  - 4 Ablation of Periplakin Expression by RNA Interference
  - 5 Identification of Periplakin Interaction Partners
  - 6 Envoplakin and Periplakin Gene-Targeted Mice
  - 7 Conclusions and Open Questions
  - Acknowledgments
- Chapter Seventeen: Microtubule-Actin Cross-Linking Factor 1: Domains, Interaction Partners, and Tissue-Specific Functions
  - Abstract
  - 1 Introduction
  - 2 MACF1 Domains
  - 3 Methods to Study Plakin Domain Interactions
  - 4 Tissue-Specific Functions of MACF1 in Mammals
  - 5 Conclusions
- Chapter Eighteen: Functional and Genetic Analysis of Neuronal Isoforms of BPAG1
  - Abstract
  - 1 Introduction
  - 2 Mouse Models
  - 3 Cell Culture System
  - 4 BPAG1 Isoform Knockdown in Immortalized Cell Lines
  - 5 BPAG1 Expression Analysis
  - 6 BPAG1 Fusion Protein Constructs
  - 7 Concluding Remarks
  - Acknowledgments
- Chapter Nineteen: Functional and Genetic Analysis of Spectraplakins in Drosophila
  - Abstract
  - 1 Introduction
  - 2 Strategies to Manipulate Shot Function
  - 3 Functional Analyses of shot In Vivo
  - 4 Functional Analyses of Shot in Cell Culture
  - 5 Informatics Approaches to Shot Protein Function
  - 6 Conclusion
  - Acknowledgments
- Chapter Twenty: Functional and Genetic Analysis of VAB-10 Spectraplakin in Caenorhabditis elegans
  - Abstract
  - 1 Introduction
  - 2 Tools
  - 3 High-Throughput RNAi Screen in L4 Worms
  - 4 2D Gel on Intermediate Filaments
  - 5 Conclusion
  - Acknowledgments
Part IV: Functional and Genetic Analysis of Lamin-Associated Proteins
- Chapter Twenty-One: Tagged Chromosomal Insertion Site System: A Method to Study Lamina-Associated Chromatin
  - Abstract
  - 1 Introduction
  - 2 Design and Preparation of the TCIS System Construct
  - 3 Generation of Stable Mammalian TCIS Clonal Lines via Random Integration
  - 4 Insert Your “DNA of Interest” into a Genomic TCIS Site
  - 5 Design and Use a Customized (or Standard) EGFP-LacI Construct for TCIS Visualization
  - 6 Detect and Localize TCIS Sites by Immunofluorescence
  - 7 Applications
  - 8 Concluding Remarks
  - Acknowledgments
- Chapter Twenty-Two: Lamin-Binding Proteins in Caenorhabditis elegans
  - Abstract
  - 1 Introduction
  - 2 Mos1-Mediated Single-Copy Insertion
  - 3 CRISPR/Cas9-Mediated Genome Engineering
  - 4 DamID Analysis of LBP Interactions with Chromatin
  - 5 Chromatin Immunoprecipitation of LBPs
  - 6 Physiological Assays for LBP Function
  - 7 FRAP and FLIP Analysis
  - 8 Summary
  - Acknowledgments
- Chapter Twenty-Three: Pathologically Relevant Prelamin A Interactions with Transcription Factors
  - Abstract
  - 1 Introduction
  - 2 Prelamin A Accumulation in hMSCS
  - 3 Identification of Transcription Factors dysregulated due to Prelamin A Accumulation
  - 4 Conclusions
  - Acknowledgments
- Chapter Twenty-Four: MCLIP Detection of Novel Protein–Protein Interactions at the Nuclear Envelope
  - Abstract
  - 1 Introduction
  - 2 MCLIP Method
  - 3 Method for Isolating Nuclei from Tissue Culture Cells
  - 4 Conclusions
  - Acknowledgments
- Chapter Twenty-Five: Preparation of the Human Cytomegalovirus Nuclear Egress Complex and Associated Proteins
  - Abstract
  - 1 Introduction
  - 2 Sample Preparation for Isolation of the NEC
  - 3 Concluding Remarks
  - Acknowledgments
Author Index
Subject Index