Cell Polarity Signaling: Methods and Protocols (Methods in Molecular Biology, 2438)

Preface
Contents
Contributors
Chapter 1: Use of Fluorescence Recovery After Photobleaching (FRAP) to Measure In Vivo Dynamics of Cell Junction-Associated Po...
1 Introduction
1.1 The Use of Fluorescent Proteins in FRAP
1.2 FRAP on In Vivo Tissue Samples
1.3 FRAP on Planar Polarity Proteins
2 Materials
2.1 Materials for Mounting Live Pupae for Imaging of Pupal Wings
2.2 Microscope Equipment
2.3 Software for Analysis of FRAP Raw Data
3 Methods
3.1 Collecting and Staging Drosophila melanogaster White Prepupae
3.2 Exposing the Drosophila Pupal Wing for Imaging
3.3 Mounting the Drosophila Pupae on an Imaging Dish
3.3.1 Mounting Method 1
3.3.2 Mounting Method 2
3.4 Live-Imaging Settings
3.5 Optimizing the FRAP Imaging Method
3.5.1 Choosing ROI Size, Shape, and Number
3.5.2 Selecting Control Unbleached Regions and Background Regions
3.5.3 Choosing the Number of Pre-bleach Images to Record
3.5.4 Specifying the Scanning Conditions Required for the Initial Bleach
3.5.5 Selecting the Number of Replicate Samples
3.5.6 Summary of FRAP Settings Used for Junctionally Associated Planar Polarity Proteins in the Drosophila Pupal Wing
3.6 Performing the FRAP Experiment
3.7 FRAP Processing Method
3.7.1 FRAP Data Collecting
3.7.2 FRAP Data Normalization
3.7.3 Graph Plotting to Check the Results
3.7.4 Curve Fitting and Extraction of Summary Data
3.7.5 Comparing Curves Between Data Sets
3.7.6 Calculating Mobile and Immobile Amounts
3.7.7 (Optional) Correcting Mean Intensity Data if the Samples Have a Variable Distance from the Coverslip
3.8 Publishing FRAP Data
4 Notes
References
Chapter 2: FRET Imaging of Rho GTPase Activity with Red Fluorescent Protein-Based FRET Pairs
1 Introduction
2 Materials
2.1 Plasmid
2.2 Reagents for Ser-FRET Imaging
2.3 Reagents for FLIM-FRET Imaging
2.4 Fluorescence Microscope
3 Methods
3.1 Ser-FRET Imaging
3.1.1 Hippocampal Neuron Preparation
3.1.2 Image Acquisition
3.1.3 Image Analysis
3.2 FLIM-FRET Imaging
3.2.1 Hippocampal Slice Preparation
3.2.2 Image Acquisition
3.2.3 Image Analysis
4 Notes
References
Chapter 3: Live-Cell Total Internal Reflection Fluorescence (TIRF) Microscopy to Investigate Protein Internalization Dynamics
1 Introduction
2 Materials
3 Methods
3.1 Laser Alignment and Focusing of Through-the-Objective TIRF
3.2 Preparing the Microscope
3.3 Imaging
3.4 Data Analysis
4 Notes
References
Chapter 4: Single-Cell Single-Molecule Pull-Down (sc-SiMPull) for Detection of Protein Complexes from Embryonic Lysates
1 Introduction
2 Materials
2.1 Microfluidic Device Master Molds
2.2 Microfluidic Devices
2.3 PEG Functionalization of Devices
2.4 C. elegans Strains and Fluorescent Labeling
2.5 Microscope
2.6 Analysis Software
3 Methods
3.1 Mold Fabrication for Microfluidic Devices
3.2 SiMPull Device Fabrication
3.2.1 Making PDMS Devices
3.2.2 Cleaning Coverslips with UV Ozone Cleaner
3.2.3 Cleaning Coverslips with Piranha Solution
3.2.4 Assembling PDMS Devices
3.3 PEGylation
3.4 Antibody Functionalization
3.5 Halo Dye Labeling
3.6 Embryo Dissection
3.7 Sealing and Lysis
3.7.1 Tape Sealing Method
3.7.2 Valap Sealing Method
3.7.3 Lysis
3.7.4 Lysis Before Sealing (for Colocalization Controls/Highly Abundant Proteins)
3.8 Data Acquisition
3.9 Automated Data Analysis
3.9.1 Analysis Software Installation
3.9.2 Automated Data Processing
3.10 Post Processing and Data Visualization
3.10.1 Sort Images and Discard Artifacts: colocalization_inspector_GUI
3.10.2 Evaluate Experiment Quality and Lysis: ``spotcount_1dplotter´´
3.10.3 Determine Labeling Efficiency and Protein Colocalization
3.10.4 Stoichiometry and Photobleaching Step Counting: See Note 10
4 Notes
References
Chapter 5: Biochemical Assays to Detect Activation of Small GTPases Rho, Rac, and Cdc42 during Morphogenesis
1 Introduction
2 Materials
2.1 Bacteria Cell Culture
2.2 Mammalian Cell Culture
2.3 Embryos
2.4 GST-PRD and GST-PBD Binding Assay Buffers
2.5 Western Blotting
3 Methods
3.1 Rho and Rac/Cdc42 Assays
3.1.1 Preparation of Recombinant GST-RBD Protein
3.1.2 Preparation of Recombinant GST-PBD Fusion Protein
3.1.3 Extraction of GST-RBD
3.1.4 Extraction of GST-PBD
3.1.5 Sample Preparations for Rho Pull-Down Assays Using Mammalian Cells
3.1.6 Sample Preparations for Rac/Cdc42 Assay Using Mammalian Cells
3.1.7 Sample Preparations for Rho/Rac/Cdc42 Assay Using Xenopus Embryos and Explants
3.1.8 GST-RBD and GST-PBD Binding Assay
3.2 Western Blot Analysis
4 Notes
References
Chapter 6: Analysis of Planar Cell Polarity Complexes by Proximity Biotinylation in Xenopus Embryos
1 Introduction
2 Materials
2.1 Xenopus Culture and Manipulation
2.2 Proximity Biotinylation and Protein Detection
3 Methods
3.1 Xenopus Embryo Culture and Microinjections
3.2 Biotinylation
3.3 Immunoprecipitation
3.4 Immunoblotting
3.5 Enhanced Chemiluminescence
4 Notes
References
Chapter 7: Regulation of Cell Polarity by Posttranslational Protein Palmitoylation
1 Introduction
2 Materials
2.1 Cell Culture Medium and Transfection Reagent
2.2 Metabolic Labeling and Click Reaction
2.3 Streptavidin Bead Pull-Down
2.4 Streptavidin Blot and Western Blot
2.5 Antibody
2.6 Constructs
2.7 3D Culture and Immunofluorescence
3 Methods
3.1 Chemical Proteomic Profiling of Palmitoylated Proteins
3.1.1 Metabolic Labeling
3.1.2 Protein Extraction
3.1.3 Click Reaction
3.1.4 Streptavidin Bead Pull-Down
3.1.5 Mass Spectrometry Analysis
3.2 Validation of SCRIB Palmitoylation
3.2.1 Transfection
3.2.2 Metabolic Labeling
3.2.3 Protein Extraction
3.2.4 Click Reaction
3.2.5 Streptavidin Blot
3.2.6 Western Blot
3.3 Identification of SCRIB Palmitoylation Sites
3.4 Exploration of the Mechanisms Regulating SCRIB Palmitoylation
3.4.1 ZDHHC-PAT Screen
3.4.2 ZDHHC-PAT and SCRIB Interactions by Co-IP Assay
3.4.3 ZDHHC7 Inactivation Analysis
3.4.4 ZDHHC7 Knockdown and Rescue Assay
3.5 Evaluation of SCRIB Palmitoylation in Regulating Cell Polarity
3.5.1 Culture MCF-10A Cells Under 3D Culture Conditions
3.5.2 Immunofluorescence Analysis of Apical-Basal Polarity
4 Notes
References
Chapter 8: Enrichment and Detection of Wnt Proteins from Cell Culture Media
1 Introduction
2 Materials
2.1 Preparation of Sample
2.2 Wnt Precipitation Using BS
2.3 Immunoblotting to Detect BS-Precipitated Wnt Proteins
3 Methods
3.1 Preparation of Sample (See Note 5)
3.2 Wnt Precipitation Using BS
3.3 Immunoblotting to Detect BS-Precipitated Wnt Proteins
4 Notes
References
Chapter 9: Using Live Imaging to Examine Early Cardiac Development in Zebrafish
1 Introduction
2 Materials
2.1 Mounting
2.2 Imaging
3 Methods
3.1 General Guidelines
Box 1 General Guidelines and Tips
3.1.1 Tailoring
3.1.2 Minimize Obstacles
3.2 Cardiomyocyte Specific Protocol
3.2.1 Preparation
3.2.2 Mounting
3.2.3 Imaging
3.2.4 Preprocessing of Images
3.2.5 Cell Tracking Analysis
4 Notes
References
Chapter 10: Imaging Planar Cell Polarity Proteins in Xenopus Neuroectoderm
1 Introduction
2 Materials
2.1 Xenopus Embryo Culture and Manipulation
2.2 PCP Protein Imaging
2.2.1 Plasmids and Antibodies
Plasmids
2.2.2 Buffers and Chemicals
2.2.3 Equipment
2.2.4 Embryo Handling Tools
3 Methods
3.1 Xenopus Embryo Culture, Microinjections, and Phenotypic Analysis
3.2 Whole Mount Immunostaining for Vangl2
3.3 Detection of Polarized Ectopic GFP-Pk3 and HA-Vangl2 Complexes in the Neural Plate (Fixed Samples)
3.3.1 Direct Fluorescence
3.3.2 Double Immunostaining
3.4 Live Imaging of Embryos Expressing Fluorescent PCP Proteins
3.5 Quantitative Analysis of PCP Based on Live or Fixed Tissue Images
4 Notes
References
Chapter 11: In Ovo Gain- and Loss-of-Function Approaches to Study Gut Morphogenesis
1 Introduction
1.1 Dorsal Mesentery as a Model to Study Gut Morphogenesis
1.2 In Ovo Electroporation of Plasmid DNA
1.3 In Ovo Electroporation of Morpholinos
1.4 In Ovo Pharmacological Targeting of Cellular Processes Through Surgical Insertion of Chemical-Soaked Resin Beads
2 Materials
2.1 In Ovo Electroporation of Plasmid DNA
2.1.1 Reagents
2.1.2 Tools
2.2 In Ovo Electroporation of Morpholinos
2.2.1 Reagents
2.2.2 Tools
2.3 In Ovo Pharmacological Targeting of Cellular Processes Through Surgical Insertion of Chemical-Soaked Resin Beads
2.3.1 Reagents
2.3.2 Tools
3 Methods
3.1 In Ovo Electroporation of Plasmid DNA
3.1.1 Egg Setup and Incubation
3.1.2 Windowing Eggs
3.1.3 DNA Electroporation
3.2 In Ovo Electroporation of Morpholinos
3.2.1 Nature of Oligos to Be Ordered
3.2.2 Making Stock Solution of Morpholino and Storage
3.2.3 Electroporating Morpholinos
3.3 In Ovo Pharmacological Targeting of Cellular Processes Through Surgical Insertion of Chemical-Soaked Resin Beads
4 Conclusion
5 Notes
References
Chapter 12: Avian Embryos as a Model to Study Vascular Development
1 Introduction
1.1 Dorsal Mesentery as a Model to Study Vascular Development
1.2 Live Imaging Using Transgenic Quails
1.3 Quail-Chick Transplantation
2 Materials
2.1 Live Imaging Using Transgenic Quails
2.1.1 Reagents
2.1.2 Tools
2.2 Quail-Chick Transplantation
2.2.1 Reagents
2.2.2 Tools
3 Methods
3.1 Live Imaging Using Transgenic Quails
3.1.1 Preparation of Embryonic Quail Slices
3.1.2 Embedding Tissue Slices
3.2 Quail-Chick Transplantation
3.2.1 Preparation of Neutral Red/Agarose to Mark the Site of Engraftment
3.2.2 Preparing the Donor (Quail) Graft Tissue
3.2.3 Preparing the Host (Chicken) Embryos to Receive the Grafts
3.2.4 Transplanting
4 Conclusion
5 Notes
References
Chapter 13: BAC Recombineering and Transgenesis to Study Cell Polarity and Polarized Tissue Morphogenesis in Mice
1 Introduction
2 Materials
2.1 Media and Antibiotics for BAC Culture
2.2 Solutions, Materials, and Equipment for BAC DNA Mini Prep
2.3 Restriction Digest and Pulse Field Gel Electrophoresis (PFGE)
2.4 Electroporation of BAC DNA into Recombineering Competent Strains
2.5 BAC Recombineering with Linear Template
2.6 Purification of BAC DNA for Pronuclear Injection
2.7 Use BAC Transgenic Mice to Study Polarized Tissue Morphogenesis
3 Methods
3.1 Identify and Acquire the Desired BAC Clones
3.2 DNA Purification from BAC Clones
3.3 Restriction Digest and Pulse Field Gene Electrophoresis (PFGE) to Characterize the Purified BAC DNA
3.4 Electroporate BAC DNA into Recombineering Competent Bacterial Strains
3.5 BAC Recombineering with Linear Template
3.6 Purification of BAC DNA for Pronuclear Injection
3.7 Use BAC Transgenic Mice to Study Polarized Tissue Morphogenesis
3.7.1 Specimen Preparation
3.7.2 Whole Mount Phalloidin Staining and RIMS Clearing
3.7.3 Confocal Imaging and 3D Reconstruction
4 Notes
References
Chapter 14: Two-Photon Cell and Tissue Level Laser Ablation Methods to Study Morphogenetic Biomechanics
1 Introduction
2 Materials
2.1 Dissection Materials
2.2 Confocal Microscope Specifications
3 Methods
3.1 Before Starting the Experiment
3.2 Embryo Collection
3.3 Embryo Positioning
3.3.1 Embryo Positioning for Cell Border Ablations
3.3.2 Embryo Positioning for Tissue-Level Ablations
3.4 Imaging and Ablation Settings
3.4.1 Imaging Settings for Cell Border Ablations
3.4.2 Imaging Settings for Tissue-Level Ablations
3.5 Image Analysis
3.5.1 Image Analysis of Cell Border Ablations
3.5.2 Image Analysis of Tissue-Level Ablations
4 Notes
References
Chapter 15: Protocols for Investigating the Epithelial Properties of Cardiac Progenitor Cells in the Mouse Embryo
1 Introduction
2 Materials
2.1 Immunofluorescence on Paraffin Sections and Whole Mount Preparations of the Dorsal Pericardial Wall Epithelium
2.1.1 Whole-Embryo Paraffin Embedding
2.1.2 Paraffin-Embedded Tissue Sectioning
2.1.3 Immunofluorescence of Paraffin-Embedded Sections
2.1.4 Immunofluorescence and Image Analysis of Whole Mount DPW Epithelium
2.2 Ex Vivo Mouse Embryo Culture and Dorsal Pericardial Wall Electroporation
2.2.1 Mouse Embryo Culture
2.2.2 Mouse Embryo Electroporation
2.3 Culture and Time-Lapse Imaging of Thick Transverse Embryo Sections
2.4 Mechanical Stress Assessment Using an Epithelial Wounding Assay
3 Methods
3.1 Immunofluorescence on Paraffin Sections and Whole Mount Preparations of the Dorsal Pericardial Wall Epithelium
3.1.1 Whole-Embryo Paraffin Embedding
3.1.2 Paraffin-Embedded Tissue Sectioning
3.1.3 Immunofluorescence of Paraffin-Embedded Sections
3.1.4 Immunofluorescence and Image Analysis of Whole Mount DPW Epithelium
3.2 Ex Vivo Mouse Embryo Culture and Dorsal Pericardial Wall Electroporation
3.2.1 Mouse Embryo Collection and Ex Vivo Culture
3.2.2 Dorsal Pericardial Wall Electroporation
3.3 Culture and Time-Lapse Imaging of Thick Transverse Embryo Sections
3.4 Mechanical Stress Assessment Using an Epithelial Wounding Assay
4 Notes
References
Chapter 16: Methods to Investigate Cell Polarity of Inner Ear
1 Introduction
2 Materials
2.1 Tools
2.2 Equipment
2.3 Reagents
2.3.1 For Dissection and Fixation
2.3.2 For Paint-Filling
2.3.3 For Frozen Section
2.3.4 For Scanning Electron Microscopy (SEM)
2.3.5 For Organ Culture
2.3.6 For EdU Detection
2.3.7 For Staining
3 Methods
3.1 Dissection of Inner Ear
3.1.1 Cochlea Dissection
3.1.2 Vestibule Dissection
3.2 Paint-Filling for Gross Development of Cochlea
3.2.1 Preparation for Paint-Filling
3.2.2 Paint-Filling
3.3 Frozen Section
3.3.1 Inner Ear Sample Preparation
3.3.2 Frozen Tissue Sectioning
3.4 SEM
3.4.1 Preparation for SEM
3.4.2 SEM
3.4.3 SEM Examination and Analysis
3.5 Whole Embryo or Organ Culture
3.5.1 Preparing for Sterile Devices
3.5.2 Preparing Complex Medium of Cell Culture and Coatings
3.6 EdU Injection in Pregnant Females for Visualization of Proliferating Cells
3.6.1 Preparation of EdU Solution
3.6.2 Injecting EdU to Pregnant Mice
3.6.3 EdU Staining
3.6.4 DNA Staining
3.7 Whole Mount or Frozen Sections Antibody Staining
3.7.1 Sample Preparation
3.7.2 Immunostaining
3.7.3 Antibody Selection
3.7.4 Mounting and Confocal Image
3.7.5 Fluorescence Observation and Image Acquisition
3.8 Image Processing and Quantification
3.8.1 Superimpose Image (Merge)
3.8.2 Image Stack Z Projection
3.8.3 Quantification of Cochlea
4 Notes
References
Chapter 17: Characterization of Axon Guidance Phenotypes in Wnt/PCP Mutant Mice
1 Introduction
1.1 Axon Guidance Phenotypes
2 Materials
2.1 Genetic Mouse Models
2.2 Immunofluorescence
2.3 In Situ Hybridization
2.4 DiI Tracing
2.5 Ex Utero Electroporation
2.6 Tamoxifen-Induced Sparse Labeling of Commissural Axons
3 Methods
3.1 Obtain or Create a Mouse Model of Your Gene of Interest
3.2 Immunofluorescence
3.3 In Situ Hybridization
3.4 DiI Tracing of Open-Book Explants
3.5 Ex Utero Electroporation
3.6 Tamoxifen-Induced Sparse Labeling of Commissural Axons
4 Notes
References
Chapter 18: In Vitro Explant Assays and Cultures to Study PCP Signaling in Axon Guidance
1 Introduction
2 Materials
2.1 Open-Book Cultures
2.1.1 Dissection, Culture, and Staining
2.1.2 Pharmacological Treatment
2.2 Pre- or Postcrossing Explant Cultures
2.2.1 Explant Dissection and Culture
2.2.2 Cell-Line Aggregates
2.3 Dissociated Neuronal Culture
2.3.1 Culture Preparation
2.3.2 Dunn Chamber Assay
2.3.3 Filopodia Tips and Vesicle Trafficking
2.3.4 Growth Cone-Growth Cone Interaction
3 Methods
3.1 Open-Book Culture
3.1.1 Dissection
3.1.2 Rat Tail Collagen Preparation
3.1.3 Culture
3.1.4 Fixation and Staining
3.1.5 Pharmacologic Treatment
3.1.6 Quantification of the Phenotype
3.2 Explant Culture
3.2.1 Explant Dissection
3.2.2 Culture Setting
3.2.3 Fixation and Staining
3.2.4 Cell-Line Aggregates
3.3 Dissociated Neuronal Culture
3.3.1 Dissociation and Culture
3.3.2 Dunn Chamber Assay
3.3.3 Filopodia Tips and Vesicle Trafficking
3.3.4 Growth Cone-Growth Cone Interaction
4 Notes
References
Chapter 19: Biochemical and Cellular Assays to Study Mechanisms of PCP Signaling in Axon Guidance
1 Introduction
2 Materials
2.1 Coimmunoprecipitation
2.2 Signaling Assays for PCP Pathway
2.3 Transcellular Interaction Assay
2.4 AP Binding Assay
3 Methods
3.1 Coimmunoprecipitation
3.2 Signaling Assays for PCP Pathway
3.3 Transcellular Interaction Assay
3.4 AP Binding Assay
4 Notes
References
Chapter 20: Dissection, Fixation, and Immunostaining of the Drosophila Midgut
1 Introduction
2 Materials
2.1 Generation of Mosaic Clones in Adult Drosophila Midgut (see Note 1)
2.1.1 Fly Stocks
2.1.2 Fly Food in Vials
2.1.3 Incubators
2.2 Fly Gut Dissection
2.3 Fixation
2.3.1 Heat Fixation Equipment (Fig. 2)
2.3.2 Buffers
2.4 Immunostaining
2.4.1 Buffers and Antibodies
2.4.2 Specimen Mounting
2.5 Imaging
3 Methods
3.1 Generation of Mutant Clones in the Adult Drosophila Midgut
3.1.1 Larval Heat Shock (LHS)
3.1.2 Adult Heat Shock (AHS)
3.2 Adult Fly Midgut Dissection
3.2.1 Preparation for Sample Dissection and Fixation
3.2.2 Sample Dissection
3.3 Heat Fixation
3.4 Immunostaining
3.5 Imaging
4 Notes
References
Chapter 21: In Vivo Analysis of Pathways Regulating Epithelial Polarity and Secretion Using Drosophila Salivary Glands
1 Introduction
2 Materials
2.1 Fly Stocks
2.2 Larvae Collection and SG Dissection
2.3 Fixation and Immunostaining of SGs
2.4 Food Intake Assay
3 Methods
3.1 Fly Crossings, Egg Collection, and Larval Staging
3.2 Preparation of Larvae for Dissections (See Note 6)
3.3 Dissection of SGs for Confocal Live Imaging
3.4 Confocal Live Imaging of Larval SG
3.5 Dissection and Fixation of Larval SGs for Immunostaining
3.6 Image Quantifications
3.7 Generating Movies from Time-Lapse Recordings
3.8 Food Intake Assay
4 Notes
References
Chapter 22: Imaging Epidermal Cell Rearrangement in the C. elegans Embryo
1 Introduction
2 Materials
2.1 Mounting Embryos for Imaging of Cell Rearrangement
2.1.1 Reagents
2.1.2 Equipment
2.2 4D Nomarski Imaging of Morphogenesis
2.3 Fluorescence Imaging of Morphogenesis
2.3.1 Phalloidin Staining for Analyzing Morphogenesis
2.4 Inducible Expression of Rho Family GTPase Constructs Using the NMD System
2.5 Laser Killing of Blastomeres
3 Methods
3.1 Mounting Embryos for Imaging of Cell Rearrangement
3.1.1 Agar Mounts for Imaging Morphogenesis; Modified from
3.1.2 Other Mounting Methods
3.2 4D Nomarski Imaging of Morphogenesis
3.2.1 Imaging Setup
3.2.2 Setting Up a 4D (or 5D) Acquisition Sequence
3.2.3 Viewing 4D/5D Datasets
3.2.4 Introducing Pharmacological Agents During 4D Acquisition
3.2.5 Creating Colorized Overlays on 4D Nomarski Movies
3.3 Fluorescence Imaging of Morphogenesis
3.3.1 General Considerations for 4D Fluorescence Imaging of Cell Rearrangement
3.3.2 Probes for Visualizing Morphogenesis
3.3.3 Quantifying Protrusive Behavior During Dorsal Intercalation
3.4 Inducible Expression of Rho Family GTPase Constructs Using the NMD System
3.5 Laser Killing of Blastomeres in Caenorhabditis elegans
3.6 Summary
4 Notes
References
Chapter 23: Methods for the Study of Apical Constriction During Ascidian Gastrulation
1 Introduction
1.1 Contributions of the Study of Fixed Embryos
1.2 Contribution of the Study of Live Embryos
1.3 Imaging Ascidian Embryos
2 Materials
2.1 Embryo Preparation
2.1.1 General Reagents
2.1.2 Ciona Work
2.1.3 Phallusia Work
2.2 Introducing Pharmacological Agents During 4D Acquisition
2.2.1 Phospho-Myosin Immunostaining
2.2.2 Phalloidin Actin Labeling
2.3 Analysis of Apical Constriction in Live Embryos Using Light Sheet Microscopy
3 Methods
3.1 Embryo Preparation
3.1.1 Gamete Collection
3.1.2 Egg Dechorionation
For Ciona
For Phallusia
3.1.3 Egg Microinjection
Preparation and Storage of Microinjection Needles
Preparing for Microinjection Under a Stereoscope
Preparation of a Microinjection Chamber to Use with an Inverted Microscope
Preparing for Microinjection Under an Inverted Microscope
Microinjection Under a Stereoscope or Inverted Microscope
3.1.4 Fertilization of Ascidian Eggs
Phallusia
Ciona
3.1.5 Embryo Staging
3.2 Analysis of Apical Constriction in Fixed Embryos
3.2.1 Phospho-Myosin Immunostaining
3.2.2 Preparation of Poly-l-Lysine-Coated Coverslips
3.2.3 Fluorescent Phalloidin Staining of Actin Cytoskeleton and Sample Mounting
3.3 Analysis of Apical Constriction in Live Embryos Using Light Sheet Microscopy
3.3.1 Fluorescent Reporters of Cell Membranes and Actomyosin Network and Optical Perturbation Tools
3.3.2 Preparation of the Sample Holder for Multiangle SPIM Imaging
3.3.3 Mounting and Imaging of Live Samples in a Multiangle SPIM
3.4 Pharmacological and Optogenetic Tools to Alter Cytoskeleton Activity
3.5 Morphological Measurements
3.5.1 Measuring Shape in Fixed Samples Imaged by Confocal Microscopy
3.5.2 Measuring Shape in Live Samples Imaged with Multiangle Light Sheet Microscopy
3.6 Myosin Activity Measurements
4 Notes
References
Chapter 24: Assays for Apical Constriction Using the Xenopus Model
1 Introduction
2 Materials
2.1 Obtaining and Culturing Xenopus Embryos
2.2 Microinjection of Early Xenopus Embryos
2.3 Staining of Actin and Activated Myosin II
2.4 Live Imaging of Actomyosin Dynamics During Gastrulation and Neurulation
3 Methods
3.1 Analyze Ectopic Induction of Apical Cell Constriction in the Animal Region (See Note 12)
3.1.1 Assessing Apical Constriction by Cell Morphology
3.1.2 Quantification of Apical Cell Surface Reduction
3.1.3 Visualization of Apical Cell Surface by Fluorescent Imaging
3.2 Inspect Actomyosin Cytoskeleton in Cells Undergoing Apical Constriction
3.2.1 Examine F-Actin Distribution in Fixed Samples
3.2.2 Analyze the Patterns of Activated Myosin in Fixed Samples
3.2.3 Explore Actomyosin Dynamics in Live Samples
3.3 Investigate Gene Function in Apical Constriction of Bottle Cells During Xenopus Gastrulation
3.4 Assess Apical Constriction During Neural Tube Closure
3.4.1 Preparation of Embryos for Confocal Imaging of NTC
4 Notes
References
Chapter 25: The Use of Three-Dimensional Cell Culture to Study Apicobasal Polarization and Lumen Formation
1 Introduction
2 Materials
2.1 MDCK Cell Culture
2.2 Generation of Stable Cell Lines
2.3 Immunofluorescence (IF) Method 1
2.4 IF Method 2
2.5 Imaging and Analysis
3 Methods
3.1 Generation of Stable Cell Lines
3.1.1 Lentiviral Production
3.1.2 Infection of MDCK Cells with Lentivirus
3.2 3D Overlay Culture of MDCK Cells
3.3 Immunofluorescence (IF) Method 1
3.4 Immunofluorescence (IF) Method 2 (Use for PIP Antibodies)
4 Notes
References
Chapter 26: Studying Cell Polarity Dynamics During Cancer Initiation Using Inducible 3D Organotypic Cultures
1 Introduction
2 Materials
2.1 Cell Line
2.2 Primary Cells
2.3 Lentivirus Production
2.4 Materials for Growing Cells in 3D Cultures
2.5 Immunostaining
3 Methods
3.1 Caco2 Cell Culture
3.1.1 Maintenance of Cultures
3.1.2 Infect Cells with Lentivirus
3.1.3 Growing 3D Cysts from Caco2 Cells (See Notes 4 and 5)
3.2 Primary Mouse Mammary Epithelial Cells
3.2.1 Isolation of Primary Mouse Mammary Epithelial Cells (See Note 6)
3.2.2 Infect Primary Cells with Lentivirus
3.2.3 Growing 3D Cysts from Primary Epithelial Cells (See Notes 4 and 5)
3.3 Induction of Gene Expression: Optional (See Note 7)
3.3.1 Caco2 Cysts
3.3.2 Primary Murine Organotypic Cultures
3.4 Immunostaining (See Notes 8-10)
3.4.1 Caco2 Cell Line
3.4.2 Mouse Mammary Organoids
3.5 Anticipated Results
4 Notes
References
Chapter 27: Under-Agarose Chemotaxis and Migration Assays for Dictyostelium
1 Introduction
2 Materials
2.1 For Dictyostelium Cell Growth
2.2 For Preparing Agarose Plates
2.3 Chemoattractants
2.4 Preparation of cAMP Competent Cells
2.5 Imaging
2.6 Image Analysis
3 Methods
3.1 Growth of Dictyostelium Cells for Chemotaxis Assays
3.2 Folate Under-Agarose Chemotaxis Assay
3.2.1 Folate Under-Agarose Chemotaxis: Preparation of Agarose Plates
3.2.2 Folate Under-Agarose Chemotaxis Assay: One Well Assay
3.2.3 Folate Under-Agarose Chemotaxis Assay: Two Well Assay
3.3 cAMP Under-Agarose Chemotaxis Assay
3.3.1 cAMP Under-Agarose Chemotaxis Assay: Preparation of cAMP Responsive Cells
3.3.2 cAMP Under-Agarose Chemotaxis Assay: Preparing Agarose
3.4 Imaging
3.5 Quantitative Image Analysis
3.5.1 Analysis and Measurement of Cell Speed, Directionality, and Chemotactic Index
3.5.2 Image Analysis: Cell Morphology
3.5.3 Image Analysis: Pseudopod Dynamics
4 Notes
References
Chapter 28: Mapping Asymmetry in Collective Cell Migration: Lessons from Border Cells in Drosophila Oogenesis
1 Introduction
2 Materials
2.1 Live Cell Imaging
2.1.1 Reagents and Dissection Tools
2.1.2 Equipment for Live Imaging
2.2 Tissue Immunohistochemistry
2.2.1 Reagents and Dissection Tool
2.3 Microscopy and Image Analysis
3 Methods
3.1 Time-Lapse Imaging and Analysis of Actin-Based Protrusions in Migrating Border Cell Clusters
3.2 Quantitative Analysis of Border Cell Clusters Captured by Live Cell Time-Lapse Imaging
3.2.1 Speed Calculation
3.2.2 Length of Protrusion
3.2.3 Direction of Protrusion
3.2.4 Number of Protrusions
3.2.5 Stability of Protrusion
3.3 Immunohistochemistry of Polarity Markers and Analysis of Distribution of Apical Basal Proteins
3.3.1 Immunostaining
3.3.2 Distribution of Apical Basal Polarity Proteins
4 Notes
References
Chapter 29: A Toolbox to Study Tissue Mechanics In Vivo and Ex Vivo
1 Introduction
2 Materials
2.1 Microinjection
2.1.1 Embryo Medium
2.1.2 Plasmids and mRNA
2.1.3 Microinjection Equipment
2.1.4 Immunofluorescence
2.2 AFM Measurements
2.3 Polyacrylamide Hydrogels
2.4 Neural Crest Dissection Tools and Protocol
2.5 Image and Statistical Analysis
3 Methods
3.1 Mesoderm Targeted Injections and Validation
3.1.1 Preparation for Microinjection
3.1.2 Setting Up the Microinjector
3.1.3 Microinjection
3.1.4 Confirmation of Injection Accuracy by Immunofluorescent Staining of Fibronectin
3.2 Analyze the Impact of PCP Inhibition on Mesoderm Cell Density In Vivo
3.3 Measuring the Impact of PCP Inhibition on Mesoderm Stiffening by In Vivo Atomic Force Microscopy (iAFM)
3.3.1 Initializing the Nanosurf Flex-ANA System (See Note 14)
3.3.2 Calibrating the Cantilever in the Nanosurf Flex-ANA System
3.3.3 Set Up the Parameters and Perform a Test Measurement
3.3.4 Determining Mesodermal Apparent Elasticity of Control and Dsh-DEP+ Embryos
3.3.5 In Vivo Atomic Force Microscopy (iAFM) Data Analysis and Presentation
3.4 Ex Vivo Analyses of NC Polarity Using Hydrogels
3.4.1 Glass Coating
3.4.2 Polyacrylamide Hydrogels Preparation
3.4.3 Gel Activation
3.4.4 Ex Vivo Analyses of Neural Crest Polarity
4 Notes
References
Chapter 30: Quantitative Analysis of Directional Neural Crest Cell Migration
1 Introduction
2 Materials
2.1 Embryo Injection
2.1.1 Embryo Medium
2.1.2 Microinjection
2.2 Explant Dissection and Culture
2.2.1 Dissection Tools
2.2.2 Matrix Preparation
2.2.3 Explant Culture
2.3 Imaging and Analysis
3 Methods
3.1 Microinjection to Label Neural Crest Cells
3.2 Prepare FN-Coated Dish
3.3 Neural Crest Explant Dissection
3.4 Neural Crest Explant Culture
3.5 Imaging and Analyzing Neural Crest Cell Migration
4 Notes
References
Index