Cell Polarity Signaling Methods and Protocols

This volume explores detailed methods to investigate various aspects of biology related to cell polarity, or asymmetry within a cell. Molecular, cellular, and tissue-level regulation and function as well as diseases caused by impairment of cell polarity are explored by these methods. Beginning with advanced imaging and biochemical methods, the book continues with planar cell polarity (PCP) signaling in morphogenesis in diverse developmental contexts, apicobasal (AB) cell polarity in development and diseases, as well as directional cell migration and biomechanics in cell polarity. The collection includes the usage of a wide variety of model systems and an extensive array of techniques, including genetic, imaging, biochemical, and biomechanical. Written for the highly successful Methods in Molecular Biology 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. 

1. Use of Fluorescence Recovery after Photobleaching (FRAP) to Measure In Vivo Dynamics of Cell Junction-Associated Polarity Proteins

            Samantha J. Warrington, Helen Strutt, and David Strutt

 

2. ­­­FRET Imaging of Rho GTPase Activity with Red Fluorescent Protein-Based FRET Pairs

            Bryce T. Bajar, Xinmeng Guan, Amy Lam, Michael Z. Lin, Ryohei Yasuda, Tal Laviv, and Jun Chu

 

3. Live-Cell Total Internal Reflection Fluorescence (TIRF) Microscopy to Investigate Protein Internalization Dynamics

            Tejeshwar C. Rao, Tomasz J. Nawara, and Alexa L. Mattheyses

 

4. Single-Cell Single Molecule Pull-Down (sc-SiMPull) for Detection of Protein Complexes from Embryonic Lysates

            Naomi Stolpner and Daniel J. Dickinson

 

5. Biochemical Assays to Detect Activation of Small GTPases Rho, Rac, and Cdc42 during Morphogenesis

            Mark L. Berns and Raymond Habas

 

6. Analysis of Planar Cell Polarity Complexes by Proximity Biotinylation in Xenopus Embryos

            Ilya Chuykin and Sergei Y. Sokol

 

7. Regulation of Cell Polarity by Posttranslational Protein Palmitoylation

            Baoen Chen, Carla Guarino, Abdelhalim Azzi, Hannah Erb, and Xu Wu

 

8. Enrichment and Detection of Wnt Proteins from Cell Culture Media

            Pooja Sonavane and Karl Willert

 

9. Using Live-Imaging to Examine Early Cardiac Development in Zebrafish

            Tess McCann, Rabina Shrestha, Alexis Graham, and Joshua Bloomekatz

 

10. Imaging Planar Cell Polarity Proteins in Xenopus Neuroectoderm

            Olga Ossipova, Pamela Mancini, and Sergei Y. Sokol

 

11. In Ovo Gain- and Loss-of-Function Approaches to Study Gut Morphogenesis

            Bhargav D. Sanketi and Natasza A. Kurpios

 

12. Avian Embryos as a Model to Study Vascular Development

            Bhargav D. Sanketi and Natasza A. Kurpios

 

13. BAC Recombineering and Transgenesis to Study Cell Polarity and Polarized Tissue Morphogenesis in Mice

            Deli Yu and Jianbo Wang

 

14. Two-Photon Cell and Tissue Level Laser Ablation Methods to Study Morphogenetic Biomechanics

            Abigail R. Marshall, Eirini Maniou, Dale Moulding, Nicholas D.E. Greene, Andrew J. Copp, and Gabriel L. Galea

 

15. Protocols for Investigating the Epithelial Properties of Cardiac Progenitor Cells in the Mouse Embryo

            Claudio Cortes, Christopher De Bono, Charlotte Thellier, Alexandre Francou, and Robert G. Kelly

 

16. Methods to Investigate Cell Polarity of Inner Ear

            Jihan Lyu, Xiaoqing Qian, Binjun Chen, and Dongdong Ren

 

17. Characterization of Axon Guidance Phenotypes in Wnt/PCP Mutant Mice

            Kathryn M. Miller, Pau Marfull-Oromí, and Yimin Zou

 

18. In Vitro Explant Assays and Cultures to Study PCP Signaling in Axon Guidance

            Pau Marfull-Oromí, Kathryn M. Miller, and Yimin Zou

 

19. Biochemical and Cellular Assays to Study Mechanisms of PCP Signaling in Axon Guidance

            Pau Marfull-Oromí, Kathryn M. Miller, and Yimin Zou

 

20. Dissection, Fixation, and Immunostaining of the Drosophila Midgut

            Jia Chen and Daniel St Johnston

 

21. In Vivo Analysis of Pathways Regulating Epithelial Polarity and Secretion Using Drosophila Salivary Glands

            Johanna Lattner, Marko Brankatschk, and David Flores-Benitez

 

22. Imaging Epidermal Cell Rearrangement in the C. elegans Embryo

            Jeff Hardin, Joel Serre, Ryan King, Elise Walck-Shannon, and David Reiner

 

23. Methods for the Study of Apical Constriction during Ascidian Gastrulation

            Ulla-Maj Fiúza and Patrick Lemaire

 

24. Assays for Apical Constriction Using the Xenopus Model

            Austin T. Baldwin, Ivan K. Popov, John B. Wallingford, and Chenbei Chang

 

25. The Use of 3-Dimensional Cell Culture to Study Apicobasal Polarization and Lumen Formation

            Álvaro Román-Fernández, Emma Sandilands, and David M. Bryant

 

26. Studying Cell Polarity Dynamics during Cancer Initiation Using Inducible 3D Organotypic Cultures

            Rachel Catterall, Reem Kurdieh, and Luke McCaffrey

 

27. Under-Agarose Chemotaxis and Migration Assays for Dictyostelium

            Shashi Prakash Singh and Robert H. Insall

 

28. Mapping Asymmetry in Collective Cell Migration: Lessons from Border Cells in Drosophila Oogenesis

            Martina Felix and Mohit Prasad

 

29. Targeted Injections, Atomic Force Microscopy, and Hydrogels: A Toolbox to Study Tissue Mechanics In Vivo and Ex Vivo

            Sofia Moreira, Jaime A. Espina, Joana E. Saraiva, and Elias H. Barriga

 

30. Quantitative Analysis of Directional Neural Crest Cell Migration

            Shuyi Nie