Cilia: Methods and Protocols (Methods in Molecular Biology, 2725)

✍ Scribed by Vito Mennella (editor)

• Publisher: Humana
• Year: 2023
• Tongue: English
• Leaves: 283
• Category: Library

✦ Synopsis

This volume covers the latest advancements in the study of ciliary complexity. Protocols cover genomic, proteomic, imaging, and functional analysis of different ciliated tissues and their wide applicability in cilia biology. Chapters in this book primarily focus on methods to study multiciliated cells, and discuss topics such as SARS-CoV-2 infections of human primary nasal multiciliated epithelial cells; expansion microscopy of ciliary proteins; live-imaging centriole amplification in mouse brain multiciliated cells; biophysical properties of cilia motility; and mucociliary transport device construction. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

Cutting-edge and thorough, Cilia: Methods and Protocols is a valuable resource for researchers who are interested in learning more about this developing field.

✦ Table of Contents

Preface
Contents
Contributors
Chapter 1: Combination of CRISPR-Cas9-RNP and Single-Cell RNAseq to Identify Cell State-Specific FOXJ1 Functions in the Human ...
1 Introduction
2 Materials
2.1 Cell Culture of Airway Cells from Nasal Turbinates
2.2 Invalidation with CRISPR-Cas9 RiboNucleoParticles (RNP)
2.3 Sequence Analysis of Deletions by Nanopore Sequencing
2.4 Dissociation of Air-Liquid-Interface Cell Cultures for Single-Cell RNAseq Analysis
2.5 Multiplexed Cell Suspension Preparation and Single-Cell RNAseq
2.6 Standard Analysis of scRNAseq with Cell Ranger and Seurat Pipelines
3 Methods
3.1 Cell Culture of Airway Cells from Nasal Turbinates
3.2 Invalidation with CRISPR-Cas9 RiboNucleoParticles (RNP)
3.3 Evaluation of Invalidation with Nanopore Sequencing
3.4 Dissociation of Air-Liquid-Interface Cell Cultures for Single-Cell RNAseq Analysis
3.5 Multiplexed Cell Suspension Preparation for Single-Cell RNAseq Analysis
3.6 Standard Analysis of scRNAseq with Cell Ranger and Seurat Pipelines
4 Notes
References
Chapter 2: SARS-CoV-2 Infection of Human Primary Nasal Multiciliated Epithelial Cells Grown on Air-Liquid Interface Cultures
1 Introduction
2 Materials
2.1 Air-Liquid Interface (ALI) Cultures of Human Primary Nasal Epithelial Cells (hPNEC)
2.1.1 Cellular Expansion: Submerged Cultures of hPNEC
2.1.2 Cell Storage
2.1.3 Cellular Differentiation: Air-Liquid Interface (ALI) Cultures of hPNEC
2.1.4 Analysis of Cellular Differentiation (Ciliation) at ALI
2.2 SARS-CoV-2 Infection of hPNEC ALI Cultures
2.2.1 Preparation of hPNEC ALI Cultures for Infection
2.2.2 SARS-CoV-2 Infection of hPNEC ALI Cultures
2.2.3 Prophylaxis Model of Infection
2.2.4 Treatment Model of Infection
2.3 Analysis of Viral Infection (Shedding) by Plaque Assay
2.3.1 Vero E6 Cell Culture
2.3.2 Plaque Assay
3 Methods
3.1 Air-Liquid Interface (ALI) Cultures of Human Primary Nasal Epithelial Cells (hPNEC)
3.1.1 Cellular Expansion: Submerged Cultures of hPNEC
3.1.2 Cell Storage
3.1.3 Cellular Differentiation: Air-Liquid Interface (ALI) Cultures of hPNEC
3.1.4 Analysis of Cellular Differentiation (Ciliation) at ALI
3.2 SARS-CoV-2 Infection of hPNEC ALI Cultures
3.2.1 Preparation of hPNEC ALI Cultures for Infection
3.2.2 SARS-CoV-2 Infection of hPNEC ALI Cultures
3.2.3 Prophylaxis Model of Infection
3.2.4 Treatment Model of Infection
3.3 Analysis of Viral Infection (Shedding) by Plaque Assay
3.3.1 Vero E6 Cell Culture
3.3.2 Plaque Assay
4 Notes
References
Chapter 3: A Chemically Inducible Organelle Rerouting Assay to Probe Primary Cilium Assembly, Maintenance, and Disassembly in ...
1 Introduction
2 Materials
2.1 Cell Culture and Transfection
2.2 Immunofluorescence
2.3 Immunoblotting
2.4 Recipes
2.4.1 Rapamycin Stock Solution
2.4.2 1x PBS
2.4.3 PBS-BT
2.4.4 Permeabilization Buffer
2.4.5 Fixation Solutions
2.4.6 RIPA Buffer
2.4.7 SDS-PAGE Sample Buffer
3 Methods
3.1 Generation FKBP and FRB-fusion Protein Expression Plasmids
3.2 Choice of Cell Lines and Expression Method
3.3 Validation of Rapamycin-inducible Rerouting of Centriolar Satellites to the Cell Periphery or Center
3.4 Stable Cell Line Generation Expressing the FKBP/FRB Fusion Proteins
3.5 Dissecting the Phenotypic Consequences of Centriolar Satellite Rerouting During Cilium Biogenesis and Function
3.5.1 Probing for Cilium Assembly Defects
3.5.2 Probing for Cilium Maintenance and Disassembly Defects
4 Notes
References
Chapter 4: Expansion Microscopy of Ciliary Proteins
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Immunofluorescence
2.3 Expansion Microscopy
3 Methods
3.1 Cell Preparation
3.2 Fixation and Blocking
3.3 Antibody Staining
3.4 Additional Anchoring
3.5 Gelation
3.6 Digestion
3.7 Post-digestion Fluorescence Staining
3.8 Expansion
3.9 Imaging
4 Notes
References
Chapter 5: Immunolabel-First-Expand-Later Expansion Microscopy Approach Using Stable STED Dyes
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Immunofluorescent Labeling (Pre-Expansion and Post-Expansion)
2.3 Microscopy
2.4 Tools and Equipment for Expansion
2.5 Expansion Reagents
2.6 Expansion Buffers and Gel Polymerization Mixture
3 Methods
3.1 Sample Fixation and Pre-Expansion Immunolabeling
3.2 Incubation with Acrylamide and Formaldehyde and Polymerization
3.3 Dividing Polymerized Gel to Smaller Samples and Denaturation
3.4 Post-Denaturation Immunolabeling
3.5 Expansion in dH2O
3.6 Imaging of Unexpanded Samples
3.7 Imaging of Expanded Samples
4 Notes
References
Chapter 6: Structural Analysis of Sperm Centrioles Using N-STORM
1 Introduction
2 Materials
2.1 Sperm Sample
2.2 Swim-Up Technique (See Note 1) and Snap Freezing
2.3 Immunofluorescence Staining
2.4 Primary and Secondary Antibodies (See Table 1)
2.5 3-D STORM Imaging
3 Methods
3.1 Swim-Up Technique (See Note 1) and Snap Freezing
3.2 Staining
3.3 Imaging
3.4 Centriole Orientation-Based Sperm Analysis (COSA)
3.5 Quantification
3.6 Statistical Analysis
4 Notes
References
Chapter 7: A Novel Sandwich Method for Serial Block Face SEM Imaging of Airway Multiciliated Epithelium
1 Introduction
2 Materials
2.1 Sandwich Embedding for Primary Cultures on Air-Liquid Interface Filters
2.2 Block Trimming and Mounting
2.3 Image Acquisition to 3D Visualization
3 Methods
3.1 Sandwich Embedding for Primary Cultures on Air-Liquid Interface
3.2 Block Trimming and Mounting
3.3 Image Acquisition to 3D Visualization
4 Notes
References
Chapter 8: Airway Cells 3D Reconstruction via Manual and Machine-Learning Aided Segmentation of Volume EM Datasets
1 Introduction
2 Materials
2.1 Equipment
2.2 Hardware
2.3 Software
3 Methods
3.1 Experimental Considerations Before Undertaking vEM Image Segmentation
3.1.1 Alignment of vEM Data
3.1.2 Configuration of the Wacom Tablet and Pen for Manual Segmentation on 3dMOD
3.2 Manual Segmentation with 3dMOD
3.2.1 Preparation Prior to Segmentation
3.2.2 Manual Annotation on 3dMOD
3.3 Deep-Learning-Based Segmentation Using Empanada-Napari
3.3.1 Preparation of Data Prior to Deep-Learning Training
3.3.2 Manual Annotation of Organelles for Deep Learning on Napari
3.3.3 Preparing Patches for Deep-learning Training on Empanada
3.3.4 Training a New Organelle Model on Empanada
3.3.5 Automated vEM Segmentation of Organelles Using the Trained Model
3.3.6 Data Visualization on 3dMOD
4 Notes
References
Chapter 9: Endogenous Tagging of Ciliary Genes in Human RPE1 Cells for Live-Cell Imaging
1 Introduction
2 Materials
2.1 Cell Culture and Transfection Reagents
2.2 Plasmids
2.3 PCR and CRISPR Reagents
2.4 Reagents for Live-Cell Imaging
2.5 Microscopes
3 Methods
3.1 Generation of Tetracycline-Inducible RPE1 Cell Lines Expressing Cas9 or Cas12a
3.2 HDR Donor and Guide RNA Design
3.3 Generation of HDR Donor by PCR
3.4 Generation of Knock-in Cell Lines
3.5 Live-Cell Imaging of eGFP-KIF13B
3.6 Image Analysis
3.6.1 Measurement of eGFP-KIF13B Intensity in Primary Cilia from Time-Lapse Image Sequences
3.6.2 Frequency Analysis of Accumulation of eGFP-KIF13B in Primary Cilia
3.6.3 Measurement and Analysis of Transport Velocities of eGFP-KIF13B and IFT Markers in Primary Cilia
4 Notes
References
Chapter 10: Live-Imaging Centriole Amplification in Mouse Brain Multiciliated Cells
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Live Cell Imaging
3 Methods
3.1 Cell Culture
3.2 Sample Installation Procedure
3.3 Selection of Imaging Positions
3.4 Launch Acquisition
4 Notes
References
Chapter 11: Proximity Mapping of Ciliary Proteins by BioID
1 Introduction
2 Materials
2.1 Cloning
2.2 Cell Culture
2.3 Transfection
2.4 SDS-Polyacrylamide (SDS-PAGE) Gel
2.5 Immunoblotting
2.6 Immunofluorescence
2.7 Cell Lysis and Affinity-Purification
3 Methods
3.1 Gateway Cloning
3.2 293 T-REx Flp-In Transfection
3.3 FLAG-BirA* Expression Validation
3.3.1 Immunofluorescence
3.3.2 Western Blotting
3.3.3 BioID Sample Expansion and Harvest
3.3.4 Cell Lysis and Affinity Purification
3.4 Liquid Chromatography - Mass Spectrometry (See Note 12)
3.4.1 Liquid Chromatography
3.4.2 Mass Spectrometry
3.4.3 LC-MS Data Analysis
3.4.4 Data Visualization
Visualization of Data in Cytoscape
Generating Dot Plots Using ProHits-Viz
Generating Heatmaps Using Heatmapper
4 Notes
References
Chapter 12: Affinity Purification of Intraflagellar Transport (IFT) Proteins in Mice Using Endogenous Streptavidin/FLAG Tags
1 Introduction
2 Materials
2.1 Gene Editing of Mice via CRISPR
2.2 Immunoprecipitation
2.3 In Solution Digest
3 Methods
3.1 sgRNA Design
3.2 Cloning of sgRNAs into T7- gRNA Vector
3.2.1 T7-gRNA Linearization
3.2.2 Ligation of Annealed Oligos into T7-gRNA Vector
3.2.3 Transformation of Competent Cells with the Ligation Mix and Isolation of Plasmid DNA
3.2.4 In Vitro Transcription of sgRNAs and Cas9 RNA
3.2.5 Gene-Editing of One-Cell Stage Murine Zygotes
3.3 Immunoprecipitation
3.4 Protein Precipitation
3.5 In Solution Digest
3.6 Data Analysis
4 Notes
References
Chapter 13: Primary Human Nasal Epithelial Cell Culture
1 Introduction
2 Materials
2.1 Coating of Flasks
2.2 Coating of Inserts
2.3 Media
2.3.1 Expansion Media (ex Media)
2.3.2 (Amounts Listed Are per 500 mL of Ex Basal Medium)
2.3.3 Differentiation Media (ALI Media)
2.4 Nasal Brushing
2.5 Passaging
2.6 Freezing Cells
3 Methods
3.1 Coating of Flasks
3.2 Coating of Inserts
3.3 Preparation of Media
3.3.1 Preparation of Expansion Media (Ex Media)
3.3.2 Preparation of Differentiation Media (ALI Media)
3.4 Nasal Brushing
3.5 Processing Nasal Brush
3.6 Cell Passaging
3.7 Seeding Cells in Transwell Inserts
3.8 Maintaining Cells in Transwell Inserts under ALI Conditions
3.9 Freezing Cells
3.10 Thawing Cells
3.10.1 Thawing Cells in Flask
3.10.2 Thawing Cells to Inserts
4 Notes
References
Chapter 14: BMI1 Transduction of Human Airway Epithelial Cells for Expansion of Proliferation and Differentiation
1 Introduction
2 Materials
3 Protocols
3.1 The Transfer Vector Cloning
3.2 Lentivirus Production (Table 1)
3.3 Titration
3.4 Transduction with BMI1 Lentivirus in Primary Airway Epithelial Cells
3.4.1 Expanding Primary Cells in a Collagen-Coated Flask
3.5 Cell Maintenance
3.5.1 Freezing BMI1 Cells
3.5.2 Thawing BMI1 Cells
3.6 Air-Liquid Interface Culture
4 Notes
References
Chapter 15: High-Speed Video Microscopy of Ependymal Cilia in Brain Organotypic and Cell Culture Models
1 Introduction
2 Materials
2.1 Brain Removal
2.2 Organotypic Brain Slice Preparation
2.3 Ependymal Cell Culture
2.3.1 Plate Preparation
2.3.2 Plate Seeding and Feeding
2.4 High-Speed Video Microscopy
3 Methods
3.1 Brain Removal to Facilitate Analysis
3.2 Organotypic Brain Slice Preparation
3.2.1 Preparation of Brain Tissue Prior to Sectioning
3.2.2 Vibratome Sectioning
3.3 Ependymal Cell Culture
3.3.1 Fibronectin Plate Preparation
3.3.2 Plate Seeding
3.3.3 Cell Culture Feeding
3.4 High-Speed Video Microscopy
3.5 Analyzing the Video Footage - Manual Counting
4 Notes
References
Chapter 16: Measuring Biophysical Properties of Cilia Motility from Mammalian Tissues via Quantitative Video Analysis Methods
1 Introduction
2 Materials
3 Methods
3.1 Imaging Set-Up
3.1.1 Imaging the Large-Scale Epithelium
3.1.2 Profile Approach
3.2 Image Analysis Protocols
3.2.1 Measuring CBF and Coverage in Motile Cilia
3.2.2 Measuring Cilia Synchronization with Multi-DDM
3.2.3 Quantifying Cilia Beating Pattern Parameters with a Profile View
4 Notes
References
Chapter 17: Mucociliary Transport Device Construction and Application to Study Mucociliary Clearance
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Construction of MCTDs and Collagen Coating
2.3 Microscopy
2.4 Solutions and Solution Handling
3 Methods
3.1 Gluing the Central Cylinder
3.2 Collagen Coating of MCTDs
3.3 Cell Culture
3.3.1 Cell Handling
3.3.2 Standard Culture Washing Procedure
3.4 Determination of CCT/Measurement of Transport in MCTDs
3.4.1 Setting up the Microscope
3.4.2 Standard Video Capture Procedure
3.4.3 Measurement of Transport with Diluted Native Mucus
3.4.4 Measurement of Transport Using Exogenous Mucus
3.5 Analysis
4 Notes
References
Index