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Sophie Vriz | Takeaki Ozawa



Optogenetic tools have allowed significant advances in the understanding of biological problems, particularly in the neurosciences field. Biological tools as well as optical set-ups have evolved and a wide range of probes and light-controllable modules are now available.
The aim of this book is to give a flavour of illumination strategies and imaging with an overview of the different optogenetic tools and their main applications in cell biology. Based on examples covering the different aspects of cell biology, this book provides a practical approach for using light-emitting sensors and light-driven actuators.

Table of Contents

Section Title Page Action Price
Cover Cover
Preface vii
References ix
Contents xi
Part I - Illumination strategies and imaging 1
Chapter 1 - Fast Volumetric Imaging Using Light-sheet Microscopy. Principles and Applications 3
1.1.\rIntroduction 5
1.2.\rFrom Classical Approaches to LSFM: the Question of Optical Sectioning 5
1.2.1.\rConfocal Microscopy 7
1.2.2.\rTwo-photon Microscopy 7
1.2.3.\rLight-sheet Fluorescent Microscopy 8
1.3.\rOptical Principles of Light-sheet Microscopy 10
1.3.1.\rThe Origins of LSFM 10
1.3.2.\rSpatial Resolution in LSFM 11\rBasics of Gaussian Optics.In light-sheet microscopy, a cylindrical or scanned spherical Gaussian beam is used to illuminate the ... 11\rResolution of the Light Sheet.In standard bright-field microscopy, the spatial resolution reflects the shape of the PSF (point-s... 12\rTrade-off Between the Field of View and the Axial Resolution.In light-sheet microscopy, any configuration therefore reflects a t... 13
1.4.\rBuilding the Improved LSFM: Pushing the Limits 13
1.4.1.\rIncreasing the Spatial Resolution 13
1.4.2.\rHigh-speed Volumetric Imaging 16
1.4.3.\rContrast Enhancement 16
1.4.4.\rImaging Deeper in Semi-transparent Samples 17
1.4.5.\rLight-sheet Microscopy with a Single Objective 18
1.5.\rApplication: Light-sheet Imaging of Zebrafish Brain 19
1.5.1.\rLight-sheet-based Whole-brain Functional Imaging in Zebrafish Larvae 19
1.5.2.\rWhole-brain LSFM-based Functional Imaging to Study Sensorimotor Integration in the Vertebrate Brain 20
1.6.\rLSFM in the Next Decade 22
References 22
Chapter 2 - Super-resolution Microscopy 25
2.1.\rThe Diffraction Limit 27
2.2.\rSuper-resolution via Localization Microscopy 27
2.3.\rSingle-molecule Localization in 3D 31
2.4.\rSuper-resolution via Correlation of Fluorescence Fluctuations 32
2.5.\rSuper-resolution via Optical Non-linear Effects 34
2.6.\rSuper-resolution Based on Structured Illumination 36
2.7.\rConclusion 38
Acknowledgements 38
References 39
Part II - Light-emitting Sensors 41
Chapter 3 - The Glowing Panoply of Fluorogen-based Markers for Advanced Bioimaging 43
3.1.\rIntroduction 45
3.2.\rFluorogen-based Markers Engineered from Natural Photorec 47
3.2.1.\rFlavin-binding Cyan–Green Fluorescent Proteins 48
3.2.2.\rBiliverdin-binding Far-red and Infrared Fluorescent Pr 49
3.2.3.\rBilirubin-binding Green Fluorescent Proteins 51
3.3.\rSemi-synthetic Fluorogen-based Markers 52
3.3.1.\rSemi-synthetic Fluorogen-based Protein Markers 52\rFluorogen-activating Proteins.Activating the fluores 52\rSelf-labeling Tags.Semi-synthetic fluorogen-based markers were also obtained by exploiting site-specific labeling systems such a... 55\rFluorescence-activating and Absorption-shifting Tag.Fluorescence-activating and absorption-shifting tag (FAST) is a small protei... 56
3.3.2.\rSemi-synthetic Fluorogen-based RNA Markers 57
3.4.\rConcluding Remarks 58
Acknowledgements 58
References 58
Chapter 4 - Optogenetic Reporters for Cell Biology and Neuroscience 63
4.1.\rIntroduction to Optogenetic Reporters 65
4.2.\rDesign Strategies 66
4.2.1.\rBiFC 66
4.2.2.\rDimerization-dependent FPs 66
4.2.3.\rFRET 68
4.2.4.\rEngineered Allosteric Effects 69
4.2.5.\rEnhancement of Intrinsic FP Sensitivities 71
4.3.\rApplications of Optogenetic Reporters 72
4.3.1.\rCell Cycle 72
4.3.2.\rpH Sensing 74
4.3.3.\rProgrammed Cell Death 75\rApoptosis.Apoptosis is a highly regulated cell death process, which generally occurs to maintain or modulate cell populations in... 76\rNon-apoptotic PCD.Although apoptosis is the most thoroughly studied pathway of PCD, the mechanism and function of other types of... 78
4.3.4.\rMessenger Molecules 78\rNeurotransmitters.Neurotransmitters are the chemical messengers that transmit signals across a chemical synapse from a neuron to... 79\rCalcium Ion.Ca2+ is a ubiquitous signaling ion – it regulates the activity of various proteins allosterically, acts in a wide ra... 80\rFRET-based GECIs.The first class of GECIs, cameleons, were reported by Miyawaki and co-workers in the lab of Roger Y. Tsien in 1... 80\rSingle FP-based GECIs.Single FP-based GECIs are engineered by coupling the conformational change of a Ca2+ sensing domain to mod... 82
4.3.5.\rProtein Kinases 83
4.3.6.\rMembrane Potential 86
4.4.\rConclusions and Future Directions 88
Acknowledgements 89
References 89
Part III - Light-driven Actuators 99
Chapter 5 - Light-driven Actuators: Spatiotemporal Dynamics of Cellular Signaling Processes 101
5.1.\rIntroduction 103
5.2.\rExperimental Procedures 103
5.2.1.\rPlasmid Construction 103
5.2.2.\rCell Culture 104
5.2.3.\rBioluminescence Assay 104
5.2.4.\rDish Coating 105
5.2.5.\rConfocal Laser Scanning Microscopy Imaging 105
5.2.6.\rHalf-life Evaluation of Off Kinetics 106
5.3.\rResults and Discussion 106
5.3.1.\rTuning Switch-off Kinetics and Dimerization Efficiencies 108
5.3.2.\rAssembly Method to Enhance the Performances of the Magnet System 109
5.3.3.\rKinetic Study of the CAD–Magnet System 110
5.3.4.\rOptical Control of Membrane Morphology by the CAD–Magnet System 112
5.3.5.\rThe Induction of Cell Membrane Dynamics Using CAD–Magnet 112
5.4.\rConclusion 113
References 115
Chapter 6 - Optogenetic Control of the Generation of Reactive Oxygen Species for Photoinducible Protein Inactivation and Cell Ablation 117
6.1.\rIntroduction 119
6.2.\rMolecular Mechanism of CALI 119
6.2.1.\rPhotosensitization Mechanism 120
6.2.2.\rROS Effects on Intracellular Molecules 121
6.2.3.\rHow Specific is CALI 122
6.3.\rDevelopment of CALI Agents and Their Application in Cell Biology 122
6.3.1.\rChemical-based Photosensitizers 122
6.3.2.\rGenetically Encoded Photosensitizers 125
6.3.3.\rGenetically Encoded Photosensitizers for Photodynamic Therapy 128
6.4.\rFuture Perspectives for CALI 131
References 132
Chapter 7 - Optogenetic Tools for Quantitative Biology: The Genetically Encoded PhyB–PIF Light-inducible Dimerization System and Its Application for Controlling Signal Transduction 137
7.1.\rIntroduction 139
7.2.\rLight-induced Dimerization (LID) Systems for Controlling Cell Signaling 139
7.3.\rSynthesis of the Chromophore of Phytochrome 141
7.4.\rPhyB–PIF LID system 143
7.5.\rQuantitative Manipulation of Cell Signaling by the PhyB–PIF System 143
7.6.\rConclusion 145
Acknowledgements 146
References 146
Chapter 8 - Quantitative Control of Kinase Activity with a Mathematical Model 149
8.1.\rIntroduction 151
8.2.\rExperimental Design of the PA-Akt System 153
8.2.1.\rPrinciple of the PA-Akt System 153
8.2.2.\rDesign of the Construction of the PA-Akt System 154
8.2.3.\rNotes on Light Illumination Wavelength and Strength 155
8.3.\rApplication of the PA-Akt System for Cellular Signaling Analysis 157
8.3.1.\rOptogenetic Control of the PA-Akt System 158
8.3.2.\rDissecting the Signaling Pathway by the PA-Akt System 159
8.3.3.\rSpatial Regulation of Actin Remodeling by Localized Activation of PA-Akt 159
8.3.4.\rPrediction of Light-induced Akt Activation Using a Mathematical Model 161
8.4.\rConclusion 165
References 166
Chapter 9 - Light Control of Transcription in Cells 169
9.1.\rIntroduction 171
9.2.\rLight-inducible Transcription Systems 171
9.3.\rLight-induced Oscillatory Versus Sustained Expression of Ascl1 173
9.4.\rLight-induced Oscillatory Expression of Dll1 175
9.4.1.\rDll1 Oscillations in Neurogenesis and Somitogenesis 175
9.4.2.\rCell-to-Cell Transfer of Oscillatory Information via Dll1 Oscillations 177
9.5.\rConclusion 178
References 179
Chapter 10 - Building Light-inducible Receptor Tyrosine Kinases 181
10.1.\rIntroduction 183
10.2.\rExperimental 183
10.2.1.\rMaterials 183
10.2.2.\rSample Preparation 184
10.2.3.\rMicroscope Setup 184
10.2.4.\rImage Analysis 184
10.3.\rResults and Discussion 185
10.3.1.\rScreening OptoRTKs with PHR 185
10.3.2.\rActivation of Canonical Signaling Pathways 186
10.3.3.\rFunctional Validation of Downstream Activation 189
10.3.4.\rResponses to Different Light Conditions 191
10.3.5.\rApplication of Diverse Actuators 192
10.4.\rConclusions 194
Acknowledgements 194
References 194
Chapter 11 - Mechanotransduction and Optogenetics 197
11.1.\rIntroduction 199
11.2.\rOptogenetic Regulation of Mechanotransduction at the Tissue Level 201
11.2.1.\rSmall Rho GTPases Are Key Regulators of Force Modulation in Tissues 202
11.2.2.\rHow Can a Predefined Spatial Constraint Have an Impact on Tissue Dynamics 204
11.3.\rPhotocontrol of Mechanotransduction Processes at the Cellular Level 207
11.3.1.\rLight-sensitive Increases in Contractility Can Mimic the Initiation of Cytokinesis 207
11.3.2.\rOptogenetics Regulation of Cell Migration 208\rCell Polarization During Migration Induced by Light-sensitive Small Rho GTPases.Several studies have used FRET-based biosensors ... 210\rCell Polarization During Migration Induced by Light-sensitive G-protein Coupled Receptors (GPCRs).GPCRs are inducers of cell mig... 210\rOptogenetic Probing Reveals a Different Ca2+ Pool Implicated in Cell Migration.Besides PI(3,4,5)P3 metabolism and small GTPase r... 211
11.4.\rControlling the Individual Regulators of Mechanotransduction by Optogenetics 212
11.4.1.\rOptogenetic Control of ECM Receptors, the Integrins 213
11.4.2.\rOptogenetic Control of Signalling Elements Downstream of Integrins 214
11.4.3.\rOptogenetic Control of Actin Remodelling 215
11.5.\rOptogenetics Sheds Light on the Spatiotemporal Regulation of Signalling 216
11.5.1.\rTemporal Regulation of a Signal Transduction 216
11.5.2.\rSpatial Regulation of a Signal Transduction 217
11.5.3.\rDiffusion of a Signal as a Feature of Spatial Regulation 217
Acknowledgements 218
References 218
Subject Index 221