Photoswitching Proteins Methods and Protocols

This volume provides cutting edge protocols for using optogenetic and opto-chemical techniques across a wide spectrum of applications. Chapters guide readers through methods in neuro-optogenetics, including methods to dissect local and long-range connectivity ex vivo, investigate G-protein coupled receptors, study astrocyte-neuron networks in vitro and in vivo, employ upconversion nanoparticles for transcranial optogenetic stimulation, and use Adeno-associated virus (AAV) for delivery of optogenetic constructs. At the same time, this book covers a range of methods for non-neuronal optogenetics, including various applications of light-oxygen-voltage domains or cryptochromes for control of nucleocytoplasmic transport, transgene expression and chromatin recruitment. The book further presents protocols for traceless light-control of gene expression with melanopsin, light-inducible cAMP production, use of light-regulated receptor tyrosine kinases (Opto-RTKs), synthesis of light-dependent biomaterials, and highlights emerging tools at the intersection of optogenetics and CRISPR. 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.

 

Authoritative and cutting-edge, Photoswitching Proteins: Methods and Protocols aims to provide a comprehensive list of methods and tools to investigate highly dynamic pathways and behaviors in cells and animals using optogenetics.

1. Studying Neuronal Function Ex Vivo using Optogenetic Stimulation and Patch Clamp

Ayla Aksoy-Aksel, Julien Genty, Martin Zeller, and Ingrid Ehrlich

 

2. Optogenetic Techniques for Probing G Protein-Coupled Receptor Signaling

Nohely Abreu and Joshua Levitz

 

3. Melanopsin for Time-Controlling Activation of Astrocyte-Neuron Networks

Sara Mederos, Candela González-Arias, and Gertrudis Perea

 

4. Near-infrared Deep Brain Stimulation in Living Mice

Shuo Chen

 

5. Lab-scale Production of Recombinant Adeno-associated Viruses (AAV) for Expression of Optogenetic Elements

Janina Haar, Chiara Krämer, and Dirk Grimm

 

6. AAV-mediated Gene Delivery to Foveal Cones

Stéphane Bertin, Elena Brazhnikova, Céline Jaillard, José-Alain Sahel, and Deniz Dalkara

 

7. Engineering Optogenetic Protein Analogs

Bei Liu, Daniel J. Marston, and Klaus M. Hahn

 

8. Optogenetic Control of Nucleocytoplasmic Protein Transport

Daniel Weis and Barbara Di Ventura

 

9. CRISPR Labeling Light-inducible

Mareike D. Hoffmann, Felix Bubeck, and Dominik Niopek

 

10. Optogenetic Control of Gene Expression using Cryptochrome 2 and a Light-activated Degron

Carmen N. Hernández-Candia and Chandra L. Tucker

 

11. Optogenetic Downregulation of Protein Levels to Control Programmed Cell Death in Mammalian Cells with a Dual Blue-Light Switch

Patrick Fischbach, Patrick Gonschorek, Julia Baaske, Jamie A. Davies, Wilfried Weber, and Matias D. Zurbriggen

 

12. Tracing Reversible Light-induced Chromatin Binding with Near-infrared Fluorescent Proteins

Anne Rademacher, Fabian Erdel, Jorge Trojanowski, and Karsten Rippe

 

13. Construction of a Multiwell Light-induction Platform for Traceless Control of Gene Expression in Mammalian Cells

Maysam Mansouri, Samson Lichtenstein, Tobias Strittmatter, Peter Buchmann, and Martin Fussenegger

 

14. Dual-activation of cAMP Production through Photo-Stimulation or Chemical Stimulation

Nyla Naim, Jeff M. Reece, Xuefeng Zhang, and Daniel L. Altschuler

 

15. Synthesis of a Light-controlled Phytochrome-based Extracellular Matrix with Reversibly Adjustable Mechanical Properties

Maximilian Hörner, Philipp Hoess, Ramona Emig, Balder Rebmann, and Wilfried Weber

 

16. Design and Application of Light-regulated Receptor Tyrosine Kinases

Stephanie Kainrath and Harald Janovjak

 

17. All-optical Miniaturized Co-culture Assay of Voltage-gated Ca²⁺ Channels

Viviana Agus and Harald Janovjak

 

18. Optogenetics and CRISPR: A New Relationship Built to Last

Jan Mathony, Mareike Daniela Hoffmann, and Dominik Niopek