Cilia: Methods and Protocols (Methods in Molecular Biology, 1454)

✍ Scribed by Peter Satir (editor), Søren Tvorup Christensen (editor)

• Publisher: Humana
• Year: 2016
• Tongue: English
• Leaves: 269
• Category: Library

✦ Synopsis

This volume is the result of an explosion of molecular-based research on Cilia, which began with the discovery of the universality of intraflagellar transport (IFT) and ciliary genomics/proteomics. The chapters in this book cover topics such as: high resolution imaging and functional characterization of sensory and primary cilia in mammalian cells and zebrafish, methods to study ciliary-mediated chemoresponse in Paramecium, and methods to study centrosomes and cilia in C. elegans and Drosophila. Written in the highly successful Methods in Molecular Biology series format, chapters include introduction 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.

Practical and cutting-edge, Cilia: Methods and Protocols is broad and covers motile, sensory, and primary cells. It is a valuable resource to anyone interested in entering the field of ciliary biology using model organisms, including flagellate algae, ciliates, planaria, nematodes, insects, zebrafish, and mammalian cells.

✦ Table of Contents

Preface
Introduction
References
Contents
Contributors
Chapter 1: Methods for Studying Ciliary Import Mechanisms
1 Introduction
2 Materials
2.1 Cell Culture and Transfection
2.2 Buffers
2.3 Microinjection
2.4 FRAP
3 Methods
3.1 Analysis of Ciliary Import by Microinjection
3.2 Analysis of Ciliary Import by Fluorescence Recovery After Photobleaching (FRAP)
4 Notes
References
Chapter 2: Targeting of ASH Domain-Containing Proteins to the Centrosome
1 Introduction
2 Materials
2.1 Materials for RNA Extraction, cDNA Preparation, PCR, and Cloning
2.2 Materials for Mammalian Cell Culture and Transfection
2.3 Materials for Immunofluo-rescence Microscopy Analysis
2.4 Materials for SDS-PAGE and Western Blot Analysis
3 Methods
3.1 RNA Extraction and cDNA Preparation
3.2 PCR and Cloning Procedures
3.3 Mammalian Cell Culture and Transfection
3.4 Immunofluo­rescence Microscopy and Image Analysis
3.5 SDS-PAGE and Western Blotting
4 Notes
References
Chapter 3: Morphological and Functional Characterization of the Ciliary Pocket by Electron and Fluorescence Microscopy
1 Introduction
2 Materials
2.1 Cells and Cell Culture Media
2.2 Buffers and Solutions for Fluorescence Microscopy
2.3 Equipment
2.4 Ligands, Antibodies, Staining Reagents, Plasmids, Conjugates, and Transfection Reagents
2.5 Electron Microscopy
2.6 Preparation of Coverslips for Immunofluo­rescence Microscopy
3 Methods
3.1 Induction of Ciliogenesis in RPE1 Cells
3.2 Induction of Ciliogenesis in NT2 Cells
3.3 Transmission Electron Microscopy (TEM) Protocol for Flat-­Embedding
3.4 Transfection of Cells with Plasmid DNA in RPE1 Cells
3.5 Transfection of Cells with Plasmid DNA in NT2 Cells
3.6 Transferrin Internalization in RPE1 Cells
3.7 Transferrin Internalization in NT2 Cells
3.8 Preparation of Cells for Immunofluorescence Microscopy in RPE1 Cells
3.9 Preparation of Cells for Immunofluorescence Microscopy in NT2 Cells
3.10 Fluorescence Microscopy and 3D Image Reconstruction in RPE1 Cells
3.11 Fluorescence Microscopy and 3D Image Reconstruction in NT2 Cells
3.12 TGFβ-1 Stimulation and Localization of TGFβ-RI to the Ciliary Pocket
3.13 Live Cell Imaging for Dynamic Analysis of Clathrin or Actin at the Ciliary Pocket
4 Notes
References
Chapter 4: Methods to Study Interactions Between Ciliogenesis and Autophagy
1 Introduction
2 Materials
2.1 Cell Cultures and Ciliary Formation
2.2 Reagents for Biochemical LC3 Flux Assay
2.3 Reagents for mCherry-
2.4 Chemicals for the Inhibition of Autophagy
3 Methods
3.1 Serum Starvation
3.2 Visualizing Cilia by Immunocyto­chemistry
3.3 Correlation of Ciliogenesis and Autophagy Measurements Using Biochemical LC3 Flux Analysis
3.4 A Complementary Method for Measuring Autophagy: mCherry-­GFP-­LC3 Fluorescent Autophagy Reporter
4 Notes
References
Chapter 5: Recombinant Reconstitution and Purification of the IFT-B Core Complex from Chlamydomonas reinhardtii
1 Introduction
2 Materials
2.1 Bacterial Strains, Plasmids, Antibiotics, and Media
2.2 Equipment
2.3 Buffers and Chemicals
3 Methods
3.1 Growth of Bacterial Cultures and Induction of Protein Expression
3.2 Cell Lysis and Extract Preparation
3.3 Ni2+-NTA Affinity Purification
3.4 Tag-Cleavage, Dialysis, and Reverse Ni2+-NTA (See Note 7)
3.5 Ion-Exchange Chromatography
3.6 Size-Exclusion Chromatography (SEC)
3.7 Reconstitution of the Nonameric IFT-B Core Complex from Purified Pentameric and Tetrameric Sub-complexes
4 Notes
References
Chapter 6: Methods for Studying Movement of Molecules Within Cilia
1 Introduction
2 Materials
2.1 Preparing Strains Expressing Fluorescent Protein-Tagged Ciliary Proteins
2.2 Sample Preparation
2.3 In Vivo Microscopy
2.4 Analyzing Protein Transport During Flagella Regeneration and Repair
2.5 Analysis of Imaging Data
3 Methods
3.1 Construction and Transformation of Expression Vectors for FP Tagging of Flagellar Proteins
3.2 Sample Preparation
3.3 In Vivo Imaging
3.4 Imaging of Protein Transport During Flagellar Growth and Repair
3.4.1 Flagellar Amputation by pH Shock
3.4.2 Generation of Long-Zero Cells
3.4.3 Generation of Ciliary Chimeras by Mating
3.5 Analysis of Transport Velocity
4 Notes
References
Chapter 7: A FRAP-Based Method for Monitoring Molecular Transport in Ciliary Photoreceptor Cells In Vivo
1 Introduction
2 Materials
3 Methods
4 Notes
References
Chapter 8: Kymographic Analysis of Transport in an Individual Neuronal Sensory Cilium in Caenorhabditis elegans
1 Introduction
2 Materials
2.1 Materials and Equipment Needed to Culture C. elegans
2.2 Mounting Worms for Microscopy
2.3 Worm Imaging
2.4 Analysis
3 Methods
3.1 Preparing NGM (Nematode Growth Media) Plates
3.2 OP50 Bacterial Culture
3.3 Nematode Culture
3.4 Preparing Slides and Mounting Worms for Imaging
3.5 Acquiring Streaming Video
3.6 Analysis of Kymographs
4 Notes
References
Chapter 9: Visualization and Manipulation of Cilia and Intraciliary Calcium in the Zebrafish Left–Right Organizer
1 Introduction
2 Materials
3 Methods
3.1 Mounting Approaches for Analyzing the Zebrafish LRO
3.1.1 Inverted Microscopes
3.1.2 Upright Microscopes
3.2 Recovery of Mounted Embryos for Analysis of Downstream Phenotypes
3.3 Direct Visualization of Cilia Motility in the LRO by DIC Videomicroscopy
3.4 Paralysis of Cilia Motility in the LRO
3.5 Simultaneous Fluorescence Imaging of Motile and Immotile LRO Cilia
3.6 Spatiotemporal Mapping of Motile and Immotile Cilia in the LRO
3.7 Altering the Ratio of Motile and Immotile Cilia in the LRO
3.8 Spatiotemporal Mapping of Intraciliary Calcium Oscillations in LRO
3.9 Suppression of Intraciliary Calcium Oscillations in LRO
3.10 Assessing for Left–Right Phenotypic Defects in Zebrafish
4 Notes
References
Chapter 10: Methods for Studying Ciliary-Mediated Chemoresponse in Paramecium
1 Introduction
2 Materials
2.1 General Equipment and Solutions for Concentrating and Working with P. tetraurelia
2.2 Materials for T-Maze Assays
2.3 Materials for Proteomics
2.4 Materials for Filter Binding Assays
2.5 Materials for Electrophysiology
3 Methods
3.1 Assays of Attraction and Repulsion
3.2 Protocol for Proteomic Analysis (Adapted from [46])
3.3 Isolating Cilia
3.4 Binding Assay Using Cilia (Adapted from [34]), for cAMP
3.5 Whole Cell Binding Assay
3.6 Electrophysi­ology Procedure (Adapted from  [10, 42])
4 Notes
References
Chapter 11: STED and STORM Superresolution Imaging of Primary Cilia
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Stock Solutions or Common Reagents for Immuno
2.3 Reagents for dSTORM Imaging
2.4 Reagents for STED Imaging
2.5 A microscopy System for STORM Imaging
2.6 A Microscopy System for STED Imaging
3 Methods
3.1 Sample Preparation for dSTORM
3.1.1 Preparation of Poly-l-Lysine Coating Glass Coverslips (See Note 1)
3.1.2 Primary Cilium Growth Upon Serum Starvation
3.1.3 Conjugation of Cy3B Fluorophore to IgG Antibody (Optional, Only Required for 2-Color dSTORM Imaging)
3.1.4 Immuno fluorescence Staining
3.2 dSTORM Imaging Procedure
3.2.1 Image Correction for dSTORM
3.3 Sample Preparation for STED Microscopy
3.3.1 Preparation of Poly-l-Lysine Coating Glass Coverslips
3.3.2 Primary Cilium Growth Upon Serum Starvation
3.3.3 Immuno
3.4 STED Imaging Procedure
3.4.1 Image Processing for STED
4 Notes
References
Chapter 12: CLEM Methods for Studying Primary Cilia
1 Introduction
2 Materials
2.1 Cells and Culture Media
2.2 Chemicals, Buffers, and Solutions
2.3 Equipment
2.4 Antibodies and Staining Reagents
2.5 Microscopy and Image Acquisition
3 Methods
3.1 Sub-culturing and Maintenance of Cells
3.2 Culture Conditions for Formation of Primary Cilia
3.3 Preparation of Cells for Immunofluorescence Microscopy
3.4 SEM Preparation
3.5 Fluorescence Microscopy
3.6 Characterization of Primary Cilia
3.7 Sputter Coating
3.8 SEM Imaging
3.9 Merging of Fluorescent and SEM images
4 Notes
References
Chapter 13: Methods for Visualization of Neuronal Cilia
1 Introduction
2 Labeling and Visualization of Neuronal Cilia In Vitro
2.1 Rationale
2.2 Materials
2.3 Method
2.4 Summary
3 Labeling and Visualization of Neuronal Cilia in Sections
3.1 Rationale
3.2 Materials
3.3 Cryosection Method
3.4 Paraffin Materials
3.5 Paraffin Method
3.6 Summary
4 Expression of Fusion Proteins for Neuronal Cilia Visualization In Vivo
5 Discussion and Conclusions
References
Chapter 14: Methods to Study Centrosomes and Cilia in Drosophila
1 Introduction
1.1 Visualization of Centrosomes and Cilia Using Fluorescence Microscope
1.2 Ultrastructure of Centrosomes and Cilia in Drosophila
2 Materials
2.1 Drosophila Stocks and Husbandry
2.2 Components for Direct Visualization and Immunostaining of Sensory Neurons
2.3 Components for Immunostaining of Testes
2.4 Components for TEM Sample Preparation
2.5 Instruments
3 Methods
3.1 Direct Visualization or Immunostaining of Centrosomes and Cilia in Sensory Neurons in the Pupae and Adult
3.2 Visualizing Directly or Immunostaining of Centrosomes and Cilia in Testes
3.3 Ultrastructure Analysis of Centrosomes and Cilia in Antenna and Testes
4 Notes
References
Chapter 15: Analysis of Axonemal Assembly During Ciliary Regeneration in Chlamydomonas
1 Introduction
2 Materials
3 Methods
3.1 Initial Deciliation
3.2 Synchronous Regeneration
3.3 Collection of Time Points
4 Notes
References
Chapter 16: Planaria as a Model System for the Analysis of Ciliary Assembly and Motility
1 Introduction
2 Materials
2.1 Propagating Planaria
2.2 RNAi Knockdowns
2.3 Electron Microscopy
3 Methods
3.1 Maintaining Planaria in the Laboratory
3.2 Generating RNAi Knockdowns
3.3 Evaluating Organismal Motility
3.4 Light Microscopic Analysis of Ciliary Assembly and Activity
3.5 Electron Microscopy of Cilia Assembly and Architecture
4 Notes
References
Index