The Nuclear Envelope: Methods and Protocols (Methods in Molecular Biology, 1411)

✍ Scribed by Sue Shackleton; Philippe Collas; Eric C. Schirmer

• Tongue: English
• Leaves: 521
• Category: Library

✦ Table of Contents

Preface
Contents
Contributors
Part I: Nuclear Envelope Isolation
Chapter 1: Isolation, Proteomic Analysis, and Microscopy Confirmation of the Liver Nuclear Envelope Proteome
1 Introduction
2 Materials
2.1 Preparation of Liver Tissue
2.1.1 Animals
2.1.2 Hardware
2.1.3 Solutions
2.2 Preparation of NEs
2.2.1 Hardware
2.2.2 Solutions
2.3 Extraction of Fractions
2.3.1 Hardware
2.3.2 Solutions
2.4 Preparation and Digestion of Proteins for MudPIT
2.4.1 Hardware
2.4.2 Solutions
2.5 Packing and Loading of Microcapillary Columns
2.5.1 Hardware
2.5.2 Solutions
2.6 Liquid Chromatography Coupled to Tandem Mass Spectrometry
2.6.1 Hardware
2.6.2 Solutions
2.7 Data Analysis
2.7.1 Hardware
2.7.2 Software
2.8 Confirmation of NE Residence and INM Localization
2.8.1 Hardware
2.8.2 Solutions
3 Methods
3.1 Preparation of Tissue (Rodent Livers)
3.2 Preparation of NEs
3.3 Extraction of Fractions
3.3.1 Optimization of Extraction Conditions
3.3.2 Salt/Detergent Extraction
3.3.3 Alkali Extraction
3.3.4 Chaotrope Extraction
3.4 Preparation and Digestion of Proteins for MudPIT
3.4.1 Endoproteinase Lys-C + Trypsin
3.4.2 Proteinase K at High pH
3.5 Packing and Loading of Microcapillary Column
3.5.1 Single-Phase Fused Silica 100 μm Microcapillary Column
3.5.2 Double-Phase Fused Silica 250 μm Microcapillary Assembly
3.5.3 Off-Line Loading and Desalting
3.5.4 Connecting 100 μm Resolutive Column with Peptide-­Loaded 250 μm Capillary
3.6 Liquid Chromatography In Line With Tandem Mass Spectrometry
3.6.1 Multidimensional Liquid Chromatography
3.6.2 Tandem Mass Spectrometry
3.7 Data Analysis
3.7.1 Searching the MS/MS Dataset
3.7.2 Assembling and Comparing Protein Lists
3.7.3 Appending Signal Peptide and Transmembrane Domain Predictions
3.7.4 Generating a List of Putative NE Transmembrane Proteins
3.8 Confirmation of NE Residence by Fluorescence Microscopy
3.8.1 Testing Tagged Fusions for NE Targeting
3.8.2 Triton Extractions Prior to Fixation
3.8.3 Digitonin Permeabilization to Determine Membrane Topology
3.8.4 Determination of Inner vs. Outer Nuclear Membrane Localization
4 Notes
References
Chapter 2: Exploring the Protein Composition of the Plant Nuclear Envelope
1 Introduction
2 Materials
2.1 Computational Identification of Plant KASH Proteins
2.2 Proteomic Identification of New Plant Nuclear Envelope and Nuclear Pore Proteins
2.2.1 Immuno
2.2.2 SDS-PAGE and Flamingo Staining
2.2.3 Plant Materials
2.2.4 Trypsin Digestion for MS Analysis
2.3 Imaging Techniques to Identify Protein–Protein Interactions at the Plant Nuclear Envelope
2.3.1 FRAP
2.3.2 apFRET
3 Methods
3.1 Computational Identification of Plant KASH Proteins
3.1.1 Setting Up “KASHFilter” Parameters
3.1.2 Setting Up “HomologyFilter” Parameters
3.1.3 Setting Up Running Parameters
3.1.4 Running DORY
3.1.5 Manual Confirmation of the Candidates
3.1.6 Improve the “Regex for KASH Tail”
3.1.7 Use DORY for Other Purposes
3.2 Proteomic Identification of New Plant Nuclear Envelope and Nuclear Pore Proteins
3.2.1 Immuno
3.2.2 SDS-PAGE and Flamingo Staining
3.2.3 In-Gel Digestion and Peptide Extraction for Mass Spectrometry
3.3 Imaging Techniques to Identify Protein–Protein Interactions at the Plant Nuclear Envelope
3.3.1 FRAP
3.3.2 apFRET
4 Notes
References
Chapter 3: High-Efficiency Isolation of Nuclear Envelope Protein Complexes from Trypanosomes
1 Introduction
2 Materials
2.1 Preparation of Frozen Trypanosome Pellets
2.2 Conjugation of Magnetic Beads
2.3 Cryomilling
2.4 Affinity Capture
3 Methods
3.1 Preparation of Frozen Trypanosome Pellets
3.2 Cryomilling Frozen Trypanosome Pellets
3.3 Conjugating Proteins to Magnetic Beads
3.4 Affinity Capture of Tagged Proteins
3.5 Downstream Analysis
4 Notes
References
Part II: Nuclear Envelope Protein Interactions, Localization, and Dynamics
Chapter 4: Superresolution Microscopy of the Nuclear Envelope and Associated Proteins
1 Introduction
1.1 3D Structured Illumination Microscopy (3D-SIM)
1.2 Single-Molecule Localization Microscopy (SMLM)
1.3 Stimulated Emission Depletion (STED) Microscopy
1.4 Recent Applications of SRM on Nuclear Envelope Components
2 Materials
2.1 Sample Preparation
2.2 3D-SIM
2.3 dSTORM and PALM
3 Methods
3.1 Sample Preparation
3.1.1 3D-SIM: Spermatocyte Spreads for Studying the Role of KASH5
3.1.2 dSTORM and PALM: Mouse Fibroblasts
3.2 Image Acquisition
3.2.1 3D-SIM
3.2.2 dSTORM and PALM
4 Notes
References
Chapter 5: Analyses of the Dynamic Properties of Nuclear Lamins by Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Correlation Spectroscopy (FCS)
1 Introduction
2 Materials
2.1 Microscope and Sample Setup for FRAP
2.2 Data Analysis for FRAP
2.3 Microscope and Sample Setup for FCS
2.4 Data Analysis for FCS
3 Methods
3.1 Cell Culture and Transfection
3.2 FRAP
3.3 Performing a Test Bleach
3.4 Performing a FRAP Experiment
3.5 FRAP Analysis
3.6 ConfoCor 2 Adjustment and FCS Measurement
3.7 LSM and FCS Measurements for Cells
3.8 Curve Fitting and Data Analysis
4 Notes
References
Chapter 6: Probing Protein Distribution Along the Nuclear Envelope In Vivo by Using Single-Point FRAP
1 Introduction
2 Materials
2.1 Microscope Components
2.2 Tissue Culture
3 Methods
3.1 Tissue Culture and Transfection
3.2 spFRAP Experiment
3.3 Bulk FRAP Experiment
3.4 Data Analysis
3.4.1 Analysis of spFRAP Data
3.4.2 Analysis of Regular FRAP Data
4 Notes
References
Chapter 7: The Use of Two-Photon FRET–FLIM to Study Protein Interactions During Nuclear Envelope Fusion In Vivo and In Vitro
1 Introduction
2 Materials
2.1 Sea Urchin Gametes
2.2 Fertilization and Fixation of Sea Urchin Eggs
2.3 Antibody–Fluorophore Conjugation
2.4 Antibody Labeling of Sea Urchin Eggs
2.5 Two-Photon FLIM of Labeled Eggs
3 Methods
3.1 Obtaining Sea Urchin Gametes
3.2 Fertilization and Fixation of Sea Urchin Eggs
3.3 Atto 488 Conjugation to F(ab′)2 Fragment
3.4 Antibody Labeling of Sea Urchin Eggs
3.5 Two-Photon FLIM of Labeled Eggs
4 Notes
References
Chapter 8: Identifying Protein-Protein Associations at the Nuclear Envelope with BioID
1 Introduction
2 Materials
2.1 Induction of Biotinylation
2.2 BioID Pulldown
2.3 Validation of Biotinylation
3 Methods
3.1 Generation of BioID Fusion Protein
3.2 Validation of BioID Fusion Protein
3.3 Stable Cell Line Generation
3.4 Induction of Biotinylation
3.5 BioID Pulldown
3.6 Validation of BioID Pulldown
4 Notes
References
Chapter 9: In Situ Detection of Interactions Between Nuclear Envelope Proteins and Partners
1 Introduction
2 Materials
2.1 Laboratory Equipment
2.2 Reagents
2.3 Antibodies
2.4 PLA Probes and Detection Reagents
2.5 Fluorescently Labeled Secondary Antibodies
3 Methods
3.1 Antibody Preparation
3.2 Cell Preparation
3.3 Blocking
3.4 Primary Antibody Incubation
3.5 Secondary Antibody Incubation
3.6 PLA: Ligation
3.7 PLA: Rolling Circle Amplification
3.8 PLA: Final Washes and Mounting
3.9 Microscope Observation and Image Analysis
4 Notes
5 Conclusion
References
Chapter 10: Methods for Single-Cell Pulse-Chase Analysis of Nuclear Components
1 Introduction
2 Materials
2.1 Cell Culture
2.2 NanoSIMS
2.3 Structured Illumination Microscopy (OMX 3D-SIM)
2.4 Confocal Microscopy of Photoactivatable Fluorescent Proteins
3 Methods
3.1 Cell Culture
3.2 NanoSIMS
3.2.1 Preparation of Stearic Acid D35 Stock Solution
3.2.2 Stearic Acid D35 Labeling and Embedding of Cells
3.2.3 BSE Imaging and NanoSIMS
3.3 Structured Illumination Microscopy (OMX 3D-SIM)
3.4 Processing of Image Data
3.5 Visualizing Nuclear Import of a Protein of Interest with Photoactivatable Green Fluorescent Protein (PA-GFP)
3.6 Data Analysis of PA-GFP-­Tagged Protein Traffic
4 Notes
References
Chapter 11: Analysis of Nuclear Lamina Proteins in Myoblast Differentiation by Functional Complementation
1 Introduction
2 Materials
2.1 Cells
2.2 Calcium Phosphate Transfection
2.3 Immunofluo-rescence Staining
2.4 siRNA Transfection
2.5 Western Blotting
3 Methods
3.1 Lentiviral Production by Calcium Phosphate Transfection
3.2 Generation of Bulk-­Transduced C2C12 Cell Populations
3.2.1 Lentiviral Infection and Selection
3.2.2 Immunofluo-rescence Staining
3.3 Functional Complementation of Myoblast Differentiation
3.3.1 siRNA-Mediated Knockdown of Endogenous Protein
3.3.2 Myoblast Differentiation Assay
3.3.3 Assessment of Myogenic Differentiation by Immunofluorescence
3.3.4 Assessment of Myogenic Differentiation and Knockdown Efficiency by Western Blotting
4 Notes
References
Chapter 12: Analysis of Meiotic Telomere Behavior in the Mouse
1 Introduction
2 Materials
2.1 Animals and Tissues
2.2 Meiotic Chromosome Spreads
2.3 Paraffin Embedding of Testes and Ovaries, Sectioning and Preparing Paraffin Samples for Immuno-­fluorescence Microscopy
2.4 Components for Immunofluo-rescence Staining
2.5 Telomere Fluorescence In Situ Hybridization Components
2.6 Electron Microscopy (EM) Components
2.7 EM In Situ Hybridization Components
2.8 EM Immunogold Components
3 Methods
3.1 Preparation of Chromosome Spreads
3.2 PFA Fixation and Paraffin Embedding of Testis and Ovary Tissue Samples
3.3 Immuno-­fluorescence Staining on Chromosome Spreads of Spermatocytes and Oocytes
3.4 Immuno-­fluorescence Staining on Paraffin Sections to Analyze Three-­Dimensionally Preserved Meiotic Nuclei
3.5 Telomere Fluorescence In Situ Hybridization in Combination with Immuno-­fluorescence Staining
3.6 Telomere In Situ Hybridization on Electron Microscopic Samples (EM-ISH)
3.7 Electron Microscopy (EM) and Immunogold EM
3.7.1 Standard EM
3.7.2 Pre-embedding Immunogold EM
4 Notes
References
Part III: Nuclear Envelope Interactions with the Cytoskeleton
Chapter 13: Identification and Validation of Putative Nesprin Variants
1 Introduction
2 Materials
3 Methods
3.1 Bioinformatics Analyses
3.2 Rapid Amplification of cDNA Ends (RACE)
3.3 Multi-­tissue RT-PCR
3.4 Alternative Splicing of Cassette Exons
4 Notes
References
Chapter 14: Detection of Diverse and High Molecular Weight Nesprin-1 and Nesprin-2 Isoforms Using Western Blotting
1 Introduction
2 Materials
2.1 Cell/Tissue Lysis
2.2 SDS-PAGE
2.3 Immunoblotting
3 Methods
3.1 Sample Preparation: From Cell Culture to Cell Lysates
3.2 Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE)
3.3 Tank (Wet) Western Blot Electro-Transfer
3.4 Membrane Blocking and Immuno-­Blotting
4 Notes
References
Chapter 15: The Use of Polyacrylamide Hydrogels to Study the Effects of Matrix Stiffness on Nuclear Envelope Properties
1 Introduction
2 Materials
3 Method
3.1 Coverslip Activation
3.2 Hydrogel Preparation
3.3 Functionalizing Hydrogels for Tissue Culture
4 Notes
References
Chapter 16: Cell Microharpooning to Study Nucleo-Cytoskeletal Coupling
1 Introduction
2 Materials
2.1 Image Acquisition and Analysis
2.2 Micro-
2.3 Cell Culture and Labeling Reagents
3 Methods
3.1 Cell Culture Preparation
3.2 Microscope Set-Up, Microharpooning, and Image Acquisition
3.3 Image Processing and Analysis
3.4 Optional Experimental Variations
4 Notes
References
Chapter 17: Wound-Healing Assays to Study Mechanisms of Nuclear Movement in Fibroblasts and Myoblasts
1 Introduction
2 Materials
2.1 General
2.2 Nuclear Movement and TAN Line Detection in Fixed Cells
2.3 Nuclear Movement (Live-Cell Phase Contrast Microscopy)
2.4 Actin Flow and TAN Line Movement (Live-Cell Fluorescent Microscopy)
2.5 Data Analysis
3 Methods
3.1 Preparation of Cells with siRNA Knockdown
3.2 Expression of Proteins in Wound Edge Cells by Microinjection
3.3 Assay of Nuclear Position in Fixed Cells
3.4 TAN Line Detection (Fixed Cells)
3.5 Nuclear Movement (Live-Cell Phase Contrast Microscopy)
3.6 Actin Flow and TAN Line Movement (Live-Cell Fluorescent Microscopy)
3.7 Data Analysis: Centrosome Orientation
3.8 Data Analysis: Analyzing Nuclear and Centrosome Positions with ImageJ
3.9 Data Analysis: Analyzing Nuclear and Centrosome Positions with Cell Plot
4 Notes
References
Chapter 18: Methods for Assessing Nuclear Rotation and Nuclear Positioning in Developing Skeletal Muscle Cells
1 Introduction
2 Materials
2.1 C2C12 Myotube Cell Culture
2.2 Cell Transfection Reagents
2.3 Live-Cell Microscopy for Nuclear Rotation Analysis
2.4 Cell Fixation and Immunofluo-rescence
2.5 Fixed Cell Microscopy for Nuclear Distribution Analysis
3 Methods
3.1 C2C12 Myotube Culture Protocols
3.2 C2C12 Transfection (Optional, See Note 5)
3.3 Live-Cell Imaging for Analysis of Nuclear Rotation
3.4 Nuclear Rotation Analysis
3.5 Cell Fixation and Immunostaining for Analysis of Nuclear Distribution
3.6 Imaging Myotubes for Analysis of Nuclear Distribution
3.7 Analysis of Nuclear Distribution
4 Notes
References
Chapter 19: Imaging Approaches to Investigate Myonuclear Positioning in Drosophila
1 Introduction
2 Materials
2.1 Imaging of Fixed Embryonic Muscles
2.1.1 Embryo Collection and Fixation (Fig. 4)
2.1.2 Embryo Staining
2.1.3 Embryo Mounting
2.1.4 Embryo Imaging
2.2 Time-Lapse Imaging of Embryonic Muscles
2.2.1 Embryo Collection and Mounting (Fig. 5)
2.2.2 Embryo Imaging
2.3 Imaging of Formalin-
2.3.1 Larvae Collection
2.3.2 Larvae Dissection and Fixation
2.3.3 Larvae Staining and Mounting
2.3.4 Heat-­Fixed Larvae
2.3.5 Larval Imaging
2.4 Fly Stocks
3 Methods
3.1 Imaging of Fixed Embryonic Muscles
3.1.1 Embryo Collection
3.1.2 Embryo Fixation
3.1.3 Embryo Staining
3.1.4 Embryo Mounting
3.1.5 Confocal Microscopy
3.1.6 Image Analysis
3.2 Time-Lapse Imaging of Embryonic Muscles
3.2.1 Embryo Collection and Mounting
3.2.2 Confocal Microscopy
3.2.3 Image Analysis
3.3 Imaging of Formalin-
3.3.1 Larval Collection (Fig. 4)
3.3.2 Larval Dissection and Fixation with Formalin
3.3.3 Larval Staining and Mounting
3.3.4 Confocal Microscopy
3.3.5 Image Analysis
3.4 Heat-­Fixed Larvae
4 Notes
References
Part IV: Nuclear Envelope-Chromatin Interactions
Chapter 20: Mapping Nuclear Lamin-Genome Interactions by Chromatin Immunoprecipitation of Nuclear Lamins
1 Introduction
2 Materials
2.1 Laboratory Equipment
2.2 Reagents
2.3 Buffers
3 Methods
3.1 Coupling of Antibody to Magnetic Dynabeads
3.2 Cross-Linking
3.3 Cell Lysis, Chromatin Fragmentation, and Chromatin Dilution
3.4 Analysis of Sonicated Chromatin Fragment Size
3.5 Immunopre
3.6 RNase Treatment, Cross-Link Reversal, and DNA Elution
3.7 DNA Purification
3.8 Real-Time Polymerase Chain Reaction (PCR) Setup
3.9 Setup for ChiP–Seq
4 Notes
References
Chapter 21: Lamin ChIP from Chromatin Prepared by Micrococcal Nuclease Digestion
1 Introduction
2 Materials
2.1 Laboratory Equipment
2.2 Reagents
2.3 Antibodies
2.4 Buffers and Solutions
3 Methods
3.1 Formaldehyde DNA–Protein Cross-Linking in Cells
3.2 Cell Lysis
3.3 Chromatin Fragmentation
3.4 Control Analysis of Chromatin Fragment Size and Lamin Solubilization
3.5 Preclearing of Chromatin
3.6 Chromatin Immunoprecipitation
3.7 Wash of the Immunoprecipitated Complexes
3.8 DNA Elution, RNase and Proteinase K Treatment, Cross-Link Reversal
3.9 Purification of ChIP DNA
3.10 Probing the Chromatin Immunoprecipitation
4 Notes
References
Chapter 22: DamID Analysis of Nuclear Organization in Caenorhabditis elegans
1 Introduction
2 Materials
2.1 Expression Plasmids for Dam Fusions
2.2 Generation and Validation of DamID Strains
2.3 Nematode Culture
2.4 Purification and Amplification of Dam-Methylated DNA
2.5 Library Preparation
2.6 DamID Quantification and Bioinformatics Analysis
3 Methods
3.1 Construction of DamID Vector
3.2 Nematode Culture
3.3 Purification and Amplification of Dam-Methylated DNA
3.4 Library Preparation
3.5 DamID Quantification and Bioinformatics Analysis
4 Notes
References
Chapter 23: The Application of DamID to Identify Peripheral Gene Sequences in Differentiated and Primary Cells
1 Introduction
2 Materials
2.1 Detection of Mycoplasma and Treatment
2.1.1 Hardware
2.1.2 Solutions and Reagents
2.2 Production of Lentivirus
2.2.1 Hardware
2.2.2 Cells and Reagents
2.3 Optimization of Transduction Conditions
2.3.1 Hardware Required for Fluorescent Visualization and Optimization of Transduction
2.3.2 Reagents and Solutions for Enhancement of Transduction
2.4 Generation and Isolation of Dam-­Methylated DNA
2.4.1 Hardware
2.4.2 Solutions and Reagents
2.5 Amplification and Purification of Dam-­Methylated DNA Fragments
2.5.1 Hardware
2.5.2 Solutions and Reagents
2.6 Deep Sequencing, Data Analysis, and Presentation
2.6.1 Hardware
2.6.2 Software
3 Methods
3.1 Detection and Treatment of Mycoplasma in Tissue Culture Cells
3.1.1 Detection
3.1.2 Elimination of Contaminating Mycoplasma
3.2 Production of Lentivirus
3.3 Determination of Transduction Conditions for Target Cell Type
3.3.1 Determination of the Optimal Concentration of Polybrene or Protamine Sulfate
3.3.2 Determination of Optimal Amounts of Lentivirus for Transduction
3.4 Generation and Isolation of Dam-­Methylated DNA
3.5 Amplification of Dam-­Methylated DNA Fragments
3.5.1 Essential Controls to Run During Preparation
3.5.2 Amplification Steps and Controls
3.5.3 Quality Testing of Amplified Dam-­Methylated DNA
3.6 Deep Sequencing, Data Analysis and Presentation
3.6.1 Construction and Deep Sequencing of DamID Libraries
3.6.2 Analysis and Presentation of DamID Data
4 Notes
References
Chapter 24: Visualizing the Spatial Relationship of the Genome with the Nuclear Envelope Using Fluorescence In Situ Hybridization
1 Introduction
2 Materials
2.1 Two-­Dimensional Fluorescence In Situ Hybridization
2.1.1 Cell Culture and Fixation
2.1.2 Slide preparation and Denaturation
2.1.3 Degenerate Oligo Primer-PCR
2.1.4 Probe Preparation and Hybridization
2.1.5 Washing 2D FISH and Visualization of Probe
2.1.6 Ki67 Antigen Marker for Proliferation in Primary Cells
2.1.7 Microscopy and Analysis
2.2 Three-­Dimensional Fluorescence In Situ Hybridization
2.2.1 Cell Culture and Fixation
2.2.2 Probe Preparation and Hybridization
2.2.3 Slide Preparation and Denaturation
2.2.4 Washing and Visualization of Probe in 3D-FISH
2.2.5 Using Ki67 as a Proliferation Marker
2.2.6 Microscopy and Analysis
2.3 FISH in Combination with Indirect Immunofluorescence
2.4 DNA Halo Preparations
3 Methods
3.1 Two-­Dimensional Fluorescence In Situ Hybridization
3.1.1 Cell Culture and Fixation
3.1.2 Slide Preparation and Denaturation
3.1.3 DOP-PCR Protocol
3.1.4 Probe Denaturation and Hybridization
3.1.5 Washing 2D-FISH Slides
3.1.6 Ki67 Antigen Marker for Proliferation in Primary Cells
3.1.7 Microscopy and Analysis
3.2 Three-­Dimensional Fluorescence In Situ Hybridization (3D-FISH)
3.2.1 Cell Culture and Fixation
3.2.2 Probe Preparation and Hybridization
3.2.3 Slide Preparation and Denaturation
3.2.4 Washing 3-D FISH
3.2.5 Using Ki67 as a Marker for Proliferation
3.2.6 Microscopy and Analysis
3.3 3D-FISH Combined with Indirect Immunofluorescence
3.4 DNA Halo Preparations
3.4.1 Measuring the Ratio of the Residual Nucleus to the Maximum Extent of the DNA Halo
3.4.2 Analyzing Positioning of Whole Chromosomes Within a DNA Halo
4 Notes
References
Chapter 25: Visualization of Genomic Loci in Living Cells with a Fluorescent CRISPR/Cas9 System
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Medium
2.3 Transfection (See Note 1)
2.4 Buffers and Solutions for Immuno-­FISH
2.5 Microscopy
2.6 Cloning
2.7 General Laboratory Equipment
3 Methods
3.1 Cloning
3.1.1 Primer Design
3.1.2 PCR and Transformation
3.2 Cell Culture
3.3 Transfection
3.3.1 Transient Transfection
3.3.2 Stably dCas9-
3.4 Immuno-FISH
4 Notes
References
Chapter 26: Methods to Monitor DNA Repair Defects and Genomic Instability in the Context of a Disrupted Nuclear Lamina
1 Introduction
2 Materials
2.1 Western Blotting Components
2.2 Components for Detection of DNA Repair Foci
2.3 Components for Neutral Comet Assay
2.4 Components for Q-FISH
3 Methods
3.1 Western Blotting for DNA Repair Factors
3.2 Formation and Resolution of DNA Repair Foci
3.3 Neutral Comet Assay
3.4 Q-FISH
4 Notes
References
Part V: Nucleo-Cytoplasmic Transport
Chapter 27: High-Resolution Scanning Electron Microscopy and Immuno-Gold Labeling of the Nuclear Lamina and Nuclear Pore Complex
1 Introduction
1.1 Field Emission Scanning Electron Microscopy
1.2 Nuclear Envelopes for feSEM
2 Materials
2.1 Obtaining Oocytes
2.2 Isolating Nuclei and Spreading NEs
2.3 Immuno-Gold Labeling NEs
2.4 Processing for feSEM
3 Methods
3.1 Obtaining Oocytes
3.2 Isolating, Placing and Spreading the Nucleus on a Silicon Chip
3.2.1 Isolation of the Nucleus and Placing onto a Silicon Chip
3.2.2 Spreading the Nuclear Envelope
3.3 Immuno-Gold Labeling
3.4 Processing for SEM
3.4.1 Osmium Tetroxide Staining and Dehydration
3.4.2 Critical Point Drying (CPD)
3.4.3 Chromium Coating
3.5 Scanning Electron Microscopy
3.5.1 Imaging Structure
3.5.2 Imaging Immuno-
3.5.3 3D SEM
4 Notes
References
Chapter 28: An In Vitro Assay to Study Targeting of Membrane Proteins to the Inner Nuclear Membrane
1 Introduction
2 Materials
2.1 Plasmids
2.2 Cell Culture and Microscopy
2.3 Extract Preparation
2.4 TEV Protease Expression
2.5 In Vitro INM Targeting Assay
3 Methods
3.1 Construction of INM Protein Reporters
3.2 Stable Tetracycline-
3.3 Preparation of Cytosolic Extract
3.4 TEV Protease Expression and Purification
3.5 The In Vitro INM Targeting Assay
3.5.1 Semi-
3.5.2 Endpoint Assay
3.5.3 Kinetic Analysis of INM Targeting by Time-Lapse Imaging
3.5.4 Cellular and Biochemical Alterations
3.6 Quantification
3.6.1 Quantification of Endpoint Assays
3.6.2 Quantification of Time-­Lapse Images
4 Notes
References
Chapter 29: Nuclear Protein Transport in Digitonin Permeabilized Cells
1 Introduction
2 Materials
2.1 Solutions
2.2 Equipment to Prepare Cell Lysates
3 Methods
3.1 Preparation of Rabbit Reticulocyte Lysate
3.2 Preparation of Lysates from Cultured Cells
3.3 Preparation of the Reaction Mix
3.4 Preparation of the Digitonin Permeabilized Cells
3.5 The Nuclear Transport Reaction
4 Notes
References
Chapter 30: Analysis of CRM1-Dependent Nuclear Export in Permeabilized Cells
1 Introduction
2 Materials
2.1 Plasmids and Cells
2.2 Buffers for Assay and Preparation of Cytosol
2.3 Buffers and Reagents for Preparation of Recombinant Transport Factors
2.4 Additional Reagents for Transport Assays
3 Methods
3.1 Preparation of Cytosol
3.2 Preparation of Recombinant Transport Factors
3.2.1 Ran
3.2.2 CRM1
3.3 Transfections and Treatment of Cells
3.4 Analysis of Transport by Fluorescence Microscopy
3.5 Analysis of Transport by Flow Cytometry
4 Notes
References
Chapter 31: SPEED Microscopy and Its Application in Nucleocytoplasmic Transport
1 Introduction
2 Materials
2.1 Preparation of Protein Cargos and Transport Receptors for Nucleocytoplasmic Transport
2.2 Preparation of the Permeabilized Cell System
2.3 SPEED Microscopy Tracking of Single Fluorescent Molecules
2.4 2D to 3D Transform Process
3 Methods
3.1 Preparation of Protein Cargos and Transport Receptors for Nucleocytoplasmic Transport
3.2 Preparation of the Permeabilized Cell System
3.3 SPEED Microscopy Tracking of Single Fluorescent Molecules
3.4 2D to 3D Transform Process
4 Notes
References
Index