Main description:

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.

Contents:

1. Generation and Analysis of Mosaic Spinal Cord Organoids Derived from Mouse Embryonic Stem Cells

Carina Jagers and Henk Roelink

2. Evaluating Hedgehog Acyltransferase Activity and Inhibition Using the Acylation-Coupled Lipophilic Induction of Polarization (Acyl-cLIP) Assay

Sebastian A. Andrei, Thomas Lanyon-Hogg, and Edward W. Tate

3. Analysis of Protein Cholesterylation by Biorthogonal Labeling

Ao Hu, Meng Zhou, and Bao-Liang Song

4. An In Situ Fluorescence Assay for Cholesterol Transporter Activity of the Patched

Wonhwa Cho, Arthur Ralko, and Ashutosh Sharma

5. Imaging of the Ciliary Cholesterol Underling the Sonic Hedgehog Signal Transduction

Tatsuo Miyamoto, Kosuke Hosoba, Silvia Natsuko Akutsu, and Shinya Matsuura

6. Studying Hedgehog Signaling during Mouse Neural Tube Development

Sandii Constable, Kevin White, Bandarigoda Nipunika Somatilaka, and Saikat Mukhopadhyay

7. Measuring and Manipulating Membrane Cholesterol for the Study of Hedgehog Signaling

Maia Kinnebrew, Kristen A. Johnson, Arun Radhakrishnan, and Rajat Rohatgi

8. The Immunofluorescence-Based Detection of Hedgehog Pathway Components in Primary Cilia of Cultured Cells

Shu Yang and Xiaoyan Zheng

9. Analysis of Dispatched Protein Processing and Sonic Hedgehog Ligand Release

Elizabeth Cleverdon, Daniel P. Stewart, and Stacey K. Ogden

10. Expression, Purification, and Structure Determination of Human PTCH1-HH-N Complexes

Xiaofeng Qi, Philip Schmiege, Leticia Esparza, and Xiaochun Li

11. Characterization of Smoothened Phosphorylation and Activation

Jianhang Jia, Yuhong Han, Yong Suk Cho, and Jin Jiang

12. Cell-Based Assays for Smoothened Ubiquitination and Sumoylation

Yuhong Han and Jin Jiang

13. Biochemical Assays to Directly Assess Smoothened Activation by a Conformationally Sensitive Nanobody

Ishan Deshpande and Aashish Manglik

14. Studies of SMOOTHENED Activation in Cell-Free and Reconstituted Systems

Isaac B. Nelson and Benjamin R. Myers

15. Rapid, Direct SMOOTHENED Activity Assays in Live Cells Using cAMP-Based Conformational Sensors

Madison F. Walker and Benjamin R. Myers

16. Light-Induced Differentiation of Forebrain Organoids by NVOC-SAG

Ryuji Misawa and Yoshiho Ikeuchi

17. NanoBRET and NanoBiT/BRET-Based Ligand Binding Assays Permit Quantitative Assessment of Small Molecule Ligand Binding to Smoothened

Pawel Kozielewicz and Gunnar Schulte

18. Measuring Smoothened (SMO)-Mediated Activation of the Gi Protein

Heng Liu and Cheng Zhang

19. Ci/Gli Phosphorylation by the Fused/Ulk Family Kinases

Yuhong Han and Jin Jiang

20. A Method for Orthotopic Transplantation of Lung Cancer in Mice

Alexandra N. Wilson, Baozhi Chen, Xin Liu, Jonathan M. Kurie, and James Kim