Hedgehog Signaling: Methods and Protocols

✍ Scribed by Xiaochun Li

• Publisher: Humana
• Year: 2021
• Tongue: English
• Leaves: 248
• Series: Methods in Molecular Biology
• Edition: 1
• Category: Library

✦ Synopsis

This detailed book explores the technical breakthroughs with biophysical and cell biological approaches that have advanced the study of molecular mechanisms underlying the generation and transduction of the hedgehog (HH) signal, indicating the important role of sterols in this pathway. Within its pages, the volume examines techniques involving several key protein components in this pathway, including HH, the signaling ligand; Dispatched, a HH secretion regulator; Patched, the receptor of HH; Smoothened, the signal transducer used to transduce the HH signal across the plasma membrane; and GLI, the transcription factor to turn on HH target genes. Written in the highly successful Methods in Molecular Biology series format, chapters feature introductions to their respective topics, lists of the necessary materials and reagents, step-by-step readily reproducible laboratory protocols, as well as tips on troubleshooting and avoiding known pitfalls. 

Authoritative and practical, Hedgehog Signaling: Methods and Protocols provides researchers in clinical and basic science with a fresh and reliable reference for analyzing HH signal transduction through cell biological, biochemical, and biophysical approaches.

✦ Table of Contents

Preface
Contents
Contributors
Chapter 1: Generation and Analysis of Mosaic Spinal Cord Organoids Derived from Mouse Embryonic Stem Cells
1 Introduction
1.1 Using Mosaic Spinal Cord Organoids to Assess Shh Morphogenesis
2 Materials
2.1 Adherent Embryonic Stem Cell Culture
2.2 Generation of Spinal Cord Organoids
2.3 Immunostaining for Neural Markers
2.4 LacZ Reporter Assay for Transcriptional Response
3 Methods
3.1 Embryonic Stem Cell Culture
3.2 Generation of Spinal Cord Organoids
3.3 Immunostaining for Neural Progenitor Markers
3.4 LacZ Reporter Assay for Transcriptional Response
3.5 Examples of Mosaic SCOs
4 Notes
References
Chapter 2: Evaluating Hedgehog Acyltransferase Activity and Inhibition Using the Acylation-coupled Lipophilic Induction of Pol...
1 Introduction
2 Materials
2.1 Production of HHAT-Enriched Membrane Fraction (P100(sol))
2.2 Acyl-cLIP Assay
3 Methods
3.1 Production of HHAT-Enriched Membrane Fraction (P100(sol))
3.2 Acyl-cLIP Assay
4 Notes
References
Chapter 3: Analysis of Protein Cholesterylation by Biorthogonal Labeling
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Cell Lysis and Click Chemistry
2.3 Sample Processing and Immunoblotting
3 Methods
3.1 Cell Setup and Transfection
3.2 Cell Harvesting, Click Chemistry, and Protein Enrichment
4 Notes
References
Chapter 4: An In Situ Fluorescence Assay for Cholesterol Transporter Activity of the Patched
Abbreviations
1 Introduction
2 Materials
2.1 Cholesterol Sensor Preparation (see Note 1)
2.2 Lipid Stock Solutions
2.3 Cell Measurements
3 Methods
3.1 Preparation of WCR-YDA: Protein Expression, Purification, and Labeling (see Note 2)
3.2 Preparation of GUVs (see Note 3)
3.3 Cholesterol Sensor Calibration Using GUVs (see Note 4)
3.4 Measurement of Cholesterol Transporter Activity of PTCH1 in PTCH1-Expressing Cells (see Note 5)
4 Notes
References
Chapter 5: Imaging of the Ciliary Cholesterol Underlying the Sonic Hedgehog Signal Transduction
1 Introduction
2 Materials
2.1 Imaging of Ciliary Cholesterol Using Filipin III
2.2 Imaging of Ciliary Cholesterol Using AcGFP1-Tagged Domain 4 of Perfringolysin O (AcGFP1-Tagged D4)
3 Methods
3.1 Imaging of Ciliary Cholesterol Using Filipin III
3.2 Imaging of Ciliary Cholesterol Using AcGFP1-Tagged D4
4 Notes
References
Chapter 6: Studying Hedgehog Signaling During Mouse Neural Tube Development
1 Introduction
2 Materials
2.1 Chemicals and Instruments
2.2 Buffers and Solutions
2.3 Primary Antibodies
2.4 Secondary Antibodies
3 Methods
3.1 Dissecting and Preparing Embryos for Cryosectioning
3.2 Cryosectioning
3.3 Paraffin Processing of Embryos
3.4 Immunofluorescence Staining of Sections
3.5 Microscopy
3.6 Quantification of Neural Progenitor Domain Sizes
3.7 Determining Changes in Enrichment of Cilia and Centrosomal Components
4 Notes
References
Chapter 7: Measuring and Manipulating Membrane Cholesterol for the Study of Hedgehog Signaling
1 Introduction
2 Materials
2.1 Materials for Expression and Purification
2.2 Materials for Cell Culture and Hedgehog Signaling Assays
3 Methods
3.1 Generation of Lipid-Binding Toxins
3.1.1 Expression Protocol
3.1.2 Purification Protocol for ALOD4 and OlyA
3.1.3 Fluorescent Labeling of ALOD4 and OlyA
3.2 Generation of Bioavailable Sterol Complexes
3.2.1 Cholesterol Complexation with Methyl-Beta Cyclodextrin (MβCD)
3.3 Altering Accessible Cholesterol and Sphingomyelin Levels for Hedgehog Signaling Assays
3.3.1 Culturing NIH/3T3 Flp-In Cells in Lipoprotein-Depleted Serum DMEM
3.3.2 Depletion of Cellular Accessible Cholesterol with ALOD4
3.3.3 Depletion of Cellular Sphingomyelin
3.3.4 Increasing Cellular Sphingomyelin
3.4 Flow Cytometry Measurement of Outer Leaflet Cholesterol, Sphingomyelin, or Cholesterol/Sphingomyelin Complexes
3.4.1 Staining Cells with Lipid-Binding Probes for Flow Cytometry
3.4.2 Flow Cytometry Measurement
3.5 Microscopy Imaging of Primary Cilia Stained with Lipid-Binding Probes
3.5.1 Staining Primary Cilia with PFO*
3.5.2 Staining Primary Cilia with OlyA
4 Notes
References
Chapter 8: The Immunofluorescence-Based Detection of Hedgehog Pathway Components in Primary Cilia of Cultured Cells
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Antibody
2.3 Equipment
3 Methods
3.1 Culture NIH 3T3 Cells
3.2 Induce Ciliary Accumulation of Smo in NIH 3T3 Cells
3.3 Immunofluorescence Labeling of Ciliary Accumulated Smo in NIH 3T3 Cells
4 Notes
References
Chapter 9: Analysis of Dispatched Protein Processing and Sonic Hedgehog Ligand Release
1 Introduction
2 Materials
3 Methods
3.1 Seeding and Transfecting Cells
3.2 SHH Protein Release into Media (see Note 3)
3.3 SHH Retention and DISP Protein Expression in Cell Lysates (see Note 4)
3.4 Western Blot of DISP and SHH Protein Samples (see Notes 5 and 6)
3.5 Seeding Cells for Secretion-Based Reporter Assay (see Notes 8 and 9)
4 Notes
References
Chapter 10: Expression, Purification, and Structure Determination of Human PTCH1-HH-N Complexes
1 Introduction
2 Materials
2.1 Protein Expression
2.2 Protein Purification and Complex Assembly
2.3 Grid Preparation and Cryo-EM Data Collection
2.4 Data Processing and Model Building
3 Methods
3.1 Protein Expression
3.2 Protein Purification and Complex Assembly
3.3 Grid Preparation and Cryo-EM Data Collection
3.4 Data Processing and Model Building
3.5 Model Building
4 Notes
References
Chapter 11: Characterization of Smoothened Phosphorylation and Activation
1 Introduction
2 Materials
2.1 Cell Culture and Transfection
2.2 Luciferase Reporter Assay
2.3 Phospho-Specific Antibody Production
2.4 GST-Smo Fusion Protein Expression and Purification
2.5 Kinase Assays
2.6 Phospho-Tag Gel
2.7 Cell Surface Staining of Drosophila S2 Cells
2.8 Cilium Staining of NIH/3T3 Cells Expressing Myc-Tagged mSmo
3 Methods
3.1 Luciferase Reporter Assay
3.1.1 S2 Cells
3.1.2 NIH/3T3 Cells
3.2 In Vitro Kinase Assay
3.2.1 Preparation of GST-Smo C-Tail Fusion
3.2.2 In Vitro Phosphorylation Assays
3.3 In Vivo Phosphorylation Assay
3.3.1 Detect Smo Phosphorylation in S2 Cells Using Anti-SmoP Antibody
3.3.2 Detect Smo Phosphorylation in NIH/3T3 Cells Using Anti-PS1 Antibody
3.3.3 Detect Smo Phosphorylation in NIH/3T3 by Phospho-Tag Gel
3.4 Cell Surface Staining Assay
3.5 Ciliary Localization Assay
4 Notes
References
Chapter 12: Cell-Based Assays for Smoothened Ubiquitination and Sumoylation
1 Introduction
2 Materials
2.1 Cell Culture and Transfection
2.2 Smo Ubiquitination and Its Regulation by Hh and Smurf
2.3 Detect Smo SUMOylation in Drosophila S2 Cells
2.4 Detect Smo SUMOylation in Mammalian NIH/3T3 Cells
3 Methods
3.1 Detect Smo Ubiquitination in Drosophila S2 Cells and Its Regulation by Hh and Smurf
3.2 Detect Smo SUMOylation in Drosophila S2 Cells
3.3 Detect Smo SUMOylation in NIH/3T3 Cells
4 Notes
References
Chapter 13: Biochemical Assays to Directly Assess Smoothened Activation by a Conformationally Sensitive Nanobody
1 Introduction
2 Materials
2.1 Expression and Purification of NbSmo8
2.2 Fluorescent Labeling of NbSmo8
2.3 Preparation of Detergent Micelles
2.4 Expression and Purification of Smoothened
2.5 FSEC Assay with Smoothened and NbSmo8
3 Methods
3.1 Expression and Purification of NbSmo8
3.2 Fluorescent Labeling of NbSmo8 with Alexa647
3.3 Preparation of Detergent Micelles
3.3.1 10% (w/v) DDM/1% (w/v) CHS
3.3.2 5% (w/v) L-MNG/0.5% (w/v) CHS
3.3.3 5% (w/v) L-MNG/2 mol% Cholesterol
3.4 Expression and Purification of Smoothened
3.5 FSEC Assay with Smoothened and NbSmo8-Alexa647
4 Notes
References
Chapter 14: Studies of SMOOTHENED Activation in Cell-Free and Reconstituted Systems
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Isolation of Crude Membrane Fractions
2.3 GTPγS Binding Assay to Measure G Protein Coupling in Crude Membrane Fractions
2.4 SMO Purification from BacMam Infected HEK293 Suspension Cells
2.5 Reconstitution of Purified SMO into Nanodiscs
3 Methods
3.1 Cell Culture and SMO Expression
3.1.1 Adherent Cells
3.1.2 Suspension Cells
3.2 Isolation of Crude Membrane Fractions
3.2.1 For Adherent Cells Grown in 10 cm Dishes
3.2.2 For Suspension Cells
3.3 GTPγS Binding Assay to Measure G Protein Coupling in Crude Membrane Fractions
3.3.1 Standard GTPγS Binding Assay Without Sterol Depletion or Repletion Studies
3.3.2 Modified GTPγS Binding Assay with Sterol Depletion or Repletion Studies
3.4 SMO Purification from HEK293 Suspension Cells
3.5 Reconstitution of Purified SMO into Nanodiscs
4 Notes
References
Chapter 15: Rapid, Direct SMOOTHENED Activity Assays in Live Cells Using cAMP-Based Conformational Sensors
1 Introduction
2 Materials
3 Methods
3.1 Culture and Transfection of HEK293FT Cells
3.1.1 Transfection Preparation
3.1.2 Transfection
3.1.3 Replating Transfected Cells
3.2 GloSensor Assay in Steady-State Mode
3.3 GloSensor Assay in Kinetic Mode
3.4 GloSensor Assay in Kinetic Mode with Ion Substitutions
4 Notes
References
Chapter 16: Light-Induced Differentiation of Forebrain Organoids by NVOC-SAG
1 Introduction
2 Materials
2.1 Culture of Human-Induced Pluripotent Stem Cells
2.2 Preparation of Forebrain Organoid
2.3 RT-PCR Analysis
2.4 Immunostaining
2.5 Solutions
2.6 Instruments
3 Methods
3.1 Synthesis of NVOC-SAG
3.2 Maintaining Human iPS Cell
3.2.1 Coating
3.2.2 Thawing
3.2.3 Passaging
3.3 Preparation of Forebrain Organoid (from Day 0 to Day 6)
3.4 Differentiation of Forebrain Organoid (from Day 6 to Day 11)
3.4.1 Light Irradiation Using a Microscope
3.4.2 Light Irradiation Using Fiber-Coupled LED
3.5 Maturation of Forebrain Organoid (from Day 11 to Day 18)
3.6 RT-PCR
3.7 Immunostaining
4 Notes
References
Chapter 17: NanoBRET and NanoBiT/BRET-Based Ligand Binding Assays Permit Quantitative Assessment of Small Molecule Ligand Bind...
Abbreviations
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Transfection Materials
2.3 Counting and Plating
2.4 Measurement Materials and Devices
2.5 Data Analysis
3 Methods
3.1 Cell Culture, Counting, Transfection, and Plating
3.2 BODIPY-Cyclopamine Saturation Binding or Competition Binding Experiments
3.3 NanoBRET or NanoBiT/BRET Measurement
3.4 Data Analysis and Graphing
4 Notes
References
Chapter 18: Measuring Smoothened (SMO)-Mediated Activation of the Gi Protein
1 Introduction
2 Materials
2.1 Smoothened Membrane Preparation
2.2 Gi Purification
2.3 [35S]-GTPγS Binding Assays
3 Methods
3.1 Smoothened Membrane Preparation
3.2 Gi Expression and Purification
3.3 GTPγS Binding Assays
4 Notes
References
Chapter 19: Ci/Gli Phosphorylation by the Fused/Ulk Family Kinases
1 Introduction
2 Materials
2.1 Cell Culture and Transfection
2.2 Phosphor Antibodies Production and Purification
2.3 Detection of Fu-Mediated Ci Phosphorylation with Phospho-Specific Antibodies in S2R+ Cells
2.4 Detection of Gli2 Phosphorylation with Phospho-Specific Antibody
2.5 GST-Ci/Gli2 Fragment Fusion Protein Expression and Purification
2.6 In Vitro Kinase Assay
2.7 Baculovirus Production and Amplification
2.8 Sf9 Cell Infection, Protein Expression, Purification
3 Methods
3.1 Hh-Conditioned Medium Preparation
3.2 Detection of Fu-Mediated Ci Phosphorylation with Phosphor-Specific Antibodies in S2R+ Cells
3.3 Detection of Gli2 Phosphorylation with Phosphor-Specific Antibodies in NIH/3T3 Cells
3.3.1 Knock Down and Reintroduce mGli2 in NIH/3T3 with Lentivirus
3.3.2 Detection of Gli2 Phosphorylation with Phosphor-Specific Antibodies
3.4 GST-Ci/Gli2 Fragment Fusion Protein Expression and Purification
3.5 Bacmid Preparation
3.6 Bacmid DNA Transfection and Baculovirus Amplification
3.7 Phosphorylation Mapping of Ci Protein Co-expressed with Active Form of Fu Using Baculovirus System
3.8 Expression and Purification of Active Form of Fu from sf9 Cells Using Baculovirus System
3.9 Expression and Purification of Ulk3 Kinase from HEK293T Cells
3.10 In Vitro Kinase Assay
3.10.1 In Vitro Kinase Assay with Fu/Ulk3 Alone
3.10.2 In Vitro Kinase Assay with Sequential Phosphorylation by Fu and CK1
3.10.3 In Vitro Kinase Assay with Sequential Phosphorylation by Ulk3 and CK1
3.10.4 32P Based Autoradiograph
3.10.5 Immunoblot with Ci Phosphor-Specific Antibodies
3.10.6 Immunoblot with pIMAGO Biotin Phosphoprotein Detection Kit
4 Notes
References
Chapter 20: A Method for Orthotopic Transplantation of Lung Cancer in Mice
1 Introduction
2 Materials
2.1 Preparation of Cells for Injection
2.2 Pre-surgical Preparation
2.3 Orthotopic Injection
2.4 Preparation of Instruments
3 Methods
3.1 Preparation of Cells for Injection
3.2 Pre-surgical Preparation
3.3 Orthotopic Injection
3.4 Removal of Wound Clips
3.5 Representative Results
4 Notes
References
Index