Fluorescent Proteins
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Table of contents
- Contentsv
- Contributorsxiii
- Prefacexix
- Chapter 1: Autofluorescent Proteins1
- I. History2
- II. Variants5
- A. Structure5
- B. Stability, Folding, and Multimerization7
- C. Spectra and Photophysical Dynamics8
- III. Practical Considerations11
- IV. Advanced FP Applications13
- A. Multiple Labeling13
- B. Dynamic Imaging14
- C. Protein-Protein Interactions15
- V. Future Directions17
- References18
- Chapter 2: Functional Fusion Proteins by Random Transposon-Based GFP Insertion23
- I. Introduction24
- II. Rationale27
- III. Methods31
- A. PCR Amplification of the Transposon31
- B. The Transposition Reaction32
- C. Transformation Requirements and Troubleshooting33
- D. E.coli Colony Selection and Growth in a 96-Well Format34
- E. Backing Up the Experiment: Making 10% Glycerol Stocks34
- F. 96-Well Mini-Preparation Purification of Plasmid DNA35
- G. Preparation of HEK 293 Cells36
- H. Transient Transfection of HEK 293 Cells in a 96-Well Format37
- I. Screening Live HEK 293 Cells for GEP Fluorescence38
- J. Removing the Selection Cassette with Restriction Digestion and Re-Ligation38
- IV. Materials40
- A. PCR Amplification of the Transposon40
- B. The Transposition Reaction40
- C. Transformation Requirements and Troubleshooting40
- D. E.coli Colony Selection and Growth in a 96-Well Format41
- E. Backing Up the Experiment: Making 10% Glycerol Stocks41
- F. 96-Well-Mini-Preparation Purification of Plasmid DNA41
- G. Preparation of HEK 293 Cells41
- H. Transient Transfection of HEK 293 Cells in a 96-Well Format41
- I. Screening Live HEK 293 Cells for GEP Fluorescence42
- J. Removing the Selection Cassette with Restriction Digestion and Re-Ligation42
- V. Discussion42
- Acknowledgments43
- References43
- Chapter 3: Fluorescent Proteins for Photoactivation Experiments45
- I. Why Use a Fluorescent Protein?46
- II. Why Use a Photoactivatable Fluorescent Protein?46
- III. Survey of Photoactivatable Fluorescent Proteins47
- A. Photoactivatable Fluorescent Proteins: Aequorea victoria GFP48
- B. Photoactivatable Fluorescent Proteins: DsRed Fluorescent Protein48
- C. Photoactivatable Fluorescent Proteins: Green-to-Red Photoconversions50
- D. Photoactivatable Fluorescent Proteins: Cyan-to-Green Photoconversion51
- E. Photoactivatable Fluorescent Proteins: Reversible51
- IV. Uses of Photoactivatable Fluorescent Proteins52
- A. Protein Dynamics52
- B. Fluorescence Pulse-Labeling.52
- C. Photoquenching Fluorescence Resonance Energy Transfer54
- D. Photoactivated Localization Microscopy55
- V. Future Directions of Photoactivatable Fluorescent Proteins58
- References59
- Chapter 4: Design and Optimization of Genetically Encoded Fluorescent Biosensors: GTPase Biosensors63
- I. Introduction64
- II. Background: Factors Influencing FRET Efficiency66
- III. Design and Cloning of Biosensors67
- IV. Validation of the Biosensor in Cell Suspensions69
- A. Expression in HEK293T Cells for Assay of Biosensors in Cell Suspension70
- B. Expression of the Biosensor in Cells72
- V. Microscopy and Imaging Considerations73
- VI. Conclusion76
- VII. Appendix I76
- A. DNA Sequence for the pTriEX-4-Biosensor Construct76
- VIII. Appendix II78
- A. Media Formulation for Ham's F-12K Phenol Red-Free78
- References79
- Chapter 5: Fast 4D Microscopy83
- I. Introduction84
- II. Fast 4D Imaging: Definition, Interest, and Limits87
- III. Points to Consider Before Working with Fast 4D Imaging Systems89
- A. Imaging Modes and Combination with Other Functionalities into a Multifunctional System89
- B. Keeping the Sampled Volume Immobile and Test for It90
- C. The Impact of Optical Blur, Noise, Aberrations, and Calibration Defects93
- D. Setting Up a Rapid 4D Acquisition99
- IV. Conclusions107
- Acknowledgments110
- References110
- Chapter 6: Single-Molecule Imaging of Fluorescent Proteins113
- I. Introduction114
- II. Instrumentation115
- III. Fluorophores118
- IV. Reducing Protein Expression Levels119
- V. Biological Preparations121
- VI. Data Analysis and Interpretation122
- VII. Future Prospects123
- References124
- Chapter 7: Counting Kinetochore Protein Numbers in Budding Yeast Using Genetically Encoded Fluoresce127
- I. Introduction128
- II. Counting Kinetochore Protein Numbers in Budding Yeast130
- A. Optimal Fluorescent Proteins134
- III. Sample Preparation134
- IV. Microscope and Image Acquisition System135
- V. Measurement of Fluorescence Signal137
- A. Characterization of the Point Spread Function of the Objective137
- B. Characterization of Fluorescence Intensity Distribution for a Kinetochore Cluster and Signal Meas138
- VI. Validation of Measurement Method140
- VII. Results142
- VIII. Discussion144
- A. The Choice of Calibration Standards for Quantitative Fluorescence Microscopy145
- B. Counting Protein Numbers from Volumes Larger than the Diffraction Limit146
- C. Sources of Error in Fluorescence Signal Measurement147
- IX. Conclusions148
- Acknowledgments149
- References149
- Chapter 8: Fluorescent Protein Applications in Plants153
- I. Introduction154
- II. Expression and Function of FPs in Plants155
- A. Gene Expression155
- B. Biosensors161
- C. Assessing Function161
- III. Imaging164
- A. Protocol for 4D Imaging169
- IV. Advanced Techniques170
- A. Spectral Imaging170
- B. Fluorescence Lifetime Imaging172
- C. Fluorescence Correlation Spectroscopy173
- V. Summary173
- Acknowledgments174
- References174
- Chapter 9: Expression and Imaging of Fluorescent Proteins in the C. elegans Gonad and Early Embryo179
- I. Introduction180
- A. The Caenorhabditis elegans Gonad and Early Embryo: A Model System for Cell and Developmental Biol180
- B. Quantitative Imaging-Based Assays Capitalize on the Rapid, Invariant Early Embryonic Cell Divisio182
- II. Fluorescent Proteins in the C. elegans Gonad and Early Embryo183
- A. Fluorescent Proteins Commonly Used in C. elegans183
- B. Engineering New Fluorescent Proteins for Expression in the C. elegans Gonad/Early Embryo: The mCh184
- III. Transgene Expression in the C. elegans Germline: Breaking the Silence187
- A. Promoter and 3' UTR Choice187
- B. Currently Available Vectors for Expression of Fluorescent Proteins in the Germline188
- IV. Constructing Fluorescent Worm Lines188
- A. Integration of Constructs by Ballistic Bombardment188
- B. Making Dual/Triple Fluorescent Marker Lines by Mating198
- C. Benefits and Challenges with Multimarker Lines201
- V. Using Fluorescent Worm Strains202
- A. Confirming Functionality of Transgenes202
- B. Available Worm Strains for Imaging in the Gonad and Early Embryo205
- C. Practical Techniques for Gonad/Embryo Imaging: Specimen Mounting and Drug Treatments205
- D. Guidelines for Live Imaging of C. elegans Embryos209
- VI. Summary210
- Acknowledgments210
- Appendix211
- Media and Supplements211
- Bombardment reagents213
- References213
- Chapter 10: Fluorescent Proteins in Zebrafish Cell and Developmental Biology219
- I. Introduction220
- II. Zebrafish Kinesin Genes in Early Development: A Cytokinetic Role for zMklp1221
- A. Cloning and Sequence Analysis of Zebrafish Kinesin cDNAs222
- B. Engineering of Expression Constructs That Encode GFP-Tagged Wild-Type and Mutant zMklp1s223
- C. In Vitro Synthesis of Capped GFP-Mklp1 mRNAs and Embryo Microinjection224
- D. Results224
- E. Future Applications and Improvements226
- III. Cell-Specific, Laser-Induced Transgene Expression in the Zebrafish Embryo: The Sema3a1 Gene in226
- A. Generation of sema3a1 Transgenic Zebrafish228
- B. Laser Induction of Transgene Expression228
- C. Detection of Axons and EGFP-Sema3a1-Myc by Immunohistochemistry229
- D. Results229
- E. Future Applications and Improvements231
- IV. Transgenic Zebrafish Models of Myc-Induced T-Cell Acute Lymphoblastic Leukemia232
- A. Generation of Zebrafish Containing the rag2:loxP-dsRED2-loxP-EGFP-mMycTransgene233
- B. Activation of the Conditional mMyc Transgene by Injection of Cre Recombinase RNA and Analysis of234
- C. Results235
- D. Future Applications and Improvements235
- V. Summary236
- Acknowledgments237
- References237
- Chapter 11: Identifying and Quantitating Neural Stem and Progenitor Cells in the Adult Brain243
- I. Introduction244
- A. Neural Stem and Progenitor Cells and Neuronal Differentiation Cascade in the Adult Brain244
- B. Adult Neurogenesis Is a Dynamic Process246
- C. Identification and Quantification of Newborn Cells in the Adult Brain246
- D. Nestin Marks Neural Stem and Progenitor Cells248
- E. Transgenic Reporter Lines for Visualizing Neural Stem and Progenitor Cells249
- F. Using Transgenic Reporter Lines to Dissect Neuronal Differentiation Cascade in the DG250
- G. Using Reporter Lines to Quantify Neural Stem and Progenitor Cells252
- II. Protocol I: Immunofluorescence Microscopy of Nestin-GFP and Nestin-CFPnuc Cells254
- A. Perfusion255
- B. Postfixation255
- C. Sectioning255
- D. Fluorescence Immunostaining256
- E. BrdU Labeling257
- F. Triple Labeling257
- III. Protocol II: The Use of Confocal Stereology to Quantify Changes in Defined Classes of Neuronal258
- A. The Fractionator Method259
- B. The Optical Disector262
- IV. Protocol III: Electron Microscopy of Nestin-GFP/CFPnuc Cells265
- A. Perfusion265
- B. Postfixation267
- C. Sectioning267
- D. 3,3'-Diaminobenzidine Immunostaining267
- E. Contrasting and Dehydration268
- F. Mounting and Embedding269
- References270
- Chapter 12: Using Fluorescent Proteins to Study mRNA Trafficking in Living Cells273
- I. Introduction274
- II. The MS2-GFP System274
- A. Designing the MS2 Fluorescent Protein Fusion275
- B. Designing the Reporter mRNA277
- III. RNA Trafficking in Fibroblasts278
- A. Mammalian Expression Systems278
- B. Fluorescent In Situ Hybridization on Fibroblasts280
- C. Visualization of RNA Movement in Living Cells284
- IV. Following RNA Trafficking in Living Yeasts287
- A. Expression in the Budding Yeast287
- B. In Situ Hybridization on Yeast Cells289
- Acknowledgments291
- References291
- Chapter 13: Visualizing mRNA Localization and Local Protein Translation in Neurons293
- I. Introduction294
- II. Visualization of RNA Transport via RNA-Binding Proteins in Neurons296
- A. Methods for Transient Transfection of Cells296
- B. Protocol for the Transfection with a DNA/CaPi Coprecipitate297
- C. Cotransfection Using More than One Plasmid303
- D. High Efficiency Transfection of Neurons by Nucleofection304
- E. Protocol for Nucleofection of Neurons305
- F. Protocol for Nucleofection with First Generation Device306
- G. Protocol for Nucleofection with the 96-Well Shuttle System307
- H. Transfection of Neurons with Lipid-Based Methods308
- III. Visualization of RNP Transport308
- A. The MS2 System to Visualize RNAs in Living Cells309
- B. Experimental Procedure311
- C. Imaging of Transfected Cells312
- D. Protocol for Live Imaging of Transfected Neurons314
- IV. Visualization of RNP Assembly and Composition316
- A. Imaging the Colocalization of trans-Acting Factors316
- B. Visualization of Direct Interactions Between trans-Acting Factors318
- V. Visualization of Interactions Between RNAs and trans-Acting Factors318
- A. Colocalization of Fluorescent Proteins with RNAs via ISH Staining319
- B. Colocalization of Fluorescent Proteins with RNAs Using the MS2 System319
- VI. Visualization of Local mRNA Translation320
- A. Protein Synthesis in Dendrites320
- B. The IRE-Based System to Detect Local Protein Synthesis322
- C. Protocol for the Detection of Local Protein Synthesis via the IRE System323
- VII. Outlook323
- Acknowledgments324
- References324
- Chapter 14: Quantitative FRAP in Analysis of Molecular Binding Dynamics In Vivo329
- I. Introduction330
- II. Rationale330
- III. Methods331
- A. Data Acquisition332
- B. Data Analysis337
- IV. Materials348
- V. Discussion348
- A. Utility of Parameters Estimated by the Models348
- B. Accuracy of Estimated Parameters349
- VI. Summary349
- Acknowledgments350
- References350
- Chapter 15: Quantitative and Qualitative Analysis of Plant Membrane Traffic Using Fluorescent Protei353
- I. Introduction354
- A. Photochemical and Biological Properties of IFPs355
- B. Vacuolar Sorting Signals in IFPs359
- II. Rationale362
- A. Need to Control Expression Level362
- B. Ratiometric Approaches to Quantify Marker Expression and Accumulation363
- C. FMDV-2A-Based Ratiometric Assays of Marker Expression and Accumulation364
- D. Quantitative Imaging of Secreted GFP Accumulation Using FMDV-2A-Based Polyproteins364
- III. Material365
- IV. Methods365
- A. Method I: Ratiometric Analysis of GFP Secretion in Populations of Cells Using YFP-2A-secG and YFP365
- B. Method II: Ratiometric Analysis of Biosynthetic Traffic in Single Cells Using nlsRFP-2A-secG and368
- V. Discussion375
- A. Quantitative Ratiometric Analaysis of secGFP Accumulation375
- B. Future Developments of 2A-Mediated Ratiometry of Membrane Traffic in Single Cells377
- VI. Summary378
- References378
- Chapter 16: Engineering FRET Constructs Using CFP and YFP381
- I. Introduction382
- II. Rationale382
- A. Optimization of the Relative Positions of CFP and YFP in a Protein or Complex382
- B. FRET Constructs Containing Long and Flexible Linkers383
- III. Methods384
- A. Optimization of the Tight Concatenation of CFP and YFP for Highly Efficient FRET384
- B. Engineering FRET Constructs Using Long and Flexible Linkers390
- References393
- Chapter 17: Fluorescence Anisotropy Imaging Microscopy for Homo-FRET in Living Cells395
- I. Introduction396
- II. Photoselection Process396
- III. Rotational Depolarization398
- IV. Experimental Measurement of Fluorescence Anisotropy Decay in Confocal Microscopy399
- V. Fluorescence Anisotropy Decay of GFP-Tagged Proteins402
- VI. Fluorescence Depolarization by Homo-FRET403
- VII. Steady-State Fluorescence Anisotropy Imaging406
- VIII. Imaging Homo-FRET by Two-Photon FAIM409
- IX. Biological Applications with GFP-Tagged Proteins410
- X. Conclusion413
- Acknowledgments413
- References413
- Chapter 18: FRET by Fluorescence Polarization Microscopy415
- I. Introduction416
- II. Measuring FRET by Polarization Microscopy418
- III. Configuration of Microscopes for AFRET419
- A. Configuring a Widefield System for Polarization Microscopy421
- B. Configuring a Laser Scanning System for Polarization Microscopy421
- IV. Calculation of Fluorescence Anisotropy424
- A. Correction for Polarization Bias in the Microscope Configuration424
- B. Correction for High NA Objective Lenses426
- V. Sample Preparation426
- A. Choice of Fluorescent Proteins426
- B. Preparing Fluorescent Protein Standards427
- C. Controls and Other Considerations428
- VI. Conclusions429
- Acknowledgments429
- References429
- Chapter 19: Bimolecular Fluorescence Complementation: Visualization of Molecular Interactions in Liv431
- I. Introduction433
- A. Roles of Protein Interactions in Regulatory Complexity434
- II. Approaches for the Investigation of Protein Interactions434
- A. Studies of Protein Interactions Using Complementation Assays435
- B. Visualization of Protein Interactions in Living Cells435
- C. Challenges for the Visualization of Protein Interactions438
- III. Bimolecular Fluorescence Complementation Analysis439
- A. Requirements for BiFC Analysis440
- B. Design of Fusion Proteins for BiFC Analysis442
- C. Instruments That Can be Used for BiFC Analysis443
- D. Effects of Fluorescent Protein Fragments on Fusion Protein Properties444
- IV. Experimental Strategies for BiFC Analysis444
- A. Design of Plasmid Vectors for Fusion Protein Expression444
- B. Strategies for Fusion Protein Expression447
- C. Controls Required for Interpretation of BiFC Experiments447
- D. Quantification of the Effciency of BiFC448
- E. Interpretation of Results from BiFC Analysis448
- V. Examples of Protein Interactions That Have Been Visualized Using the BiFC Assay449
- A. BiFC Analysis of Nuclear Proteins449
- B. BiFC Analysis of Enzyme–Substrate Complexes452
- C. BiFC Analysis of Signal Transduction Pathways452
- D. BiFC Analysis of Complex Relocalization453
- E. BiFC Analysis of Interactions Induced by Posttranslational Modifications453
- F. BiFC Analysis of Interactions on Molecular Scaffolds453
- VI. BiFC Analysis of Interactions in Different Organisms454
- VII. Screens Using the BiFC Approach455
- VIII. Analysis of Complex Dynamics Using the BiFC Approach455
- IX. Simultaneous Visualization of Several Protein Complexes456
- A. Comparison of the Distributions of Different Complexes in the Same Cell456
- B. Competition Among Mutually Exclusive Interaction Partners for Complex Formation456
- X. Experimental Strategies for Multicolor BiFC Analysis458
- A. Design of Plasmid Vectors for Multicolor BiFC Analysis458
- B. Strategies for Coexpression of Proteins for Multicolor BiFC Analysis459
- C. Quantitation of Competition Between Alternative Interaction Partners Using Multicolor BiFC Analys459
- XI. Limitations of the Multicolor BiFC Assay for Analysis of the Efficiencies of Protein Interaction460
- XII. Interaction Partners Whose Competition Has Been Visualized Using the Multicolor BiFC Assay460
- XIII. Visualization of Ubiquitin Family Peptide Conjugates in Cells461
- A. Limitations of the UbFC Assay for the Detection of Ubiquitin Family Peptide Conjugates461
- XIV. Ubiquitin Family Peptide Conjugates That Have Been Visualized Using the UbFC Assay462
- XV. Comparison of BiFC Analysis with Other Methods for the Visualization of Protein Interactions in463
- XVI. Future Opportunities and Challenges464
- Acknowledgments465
- References465
- Chapter 20: Protein-Protein Interactions Determined by Fluorescence Correlation Spectroscopy471
- I. Introduction472
- II. FCS Theory475
- A. Autocorrelation Function for One Species475
- B. Multiple Species476
- C. Triplet Contribution477
- III. Two-Color Cross-Correlation477
- IV. Protein-Protein Interactions Using FCCS and Nongenetic Labels478
- V. Protein-Protein Interactions In Vivo Using FCCS and Autofluorescent Proteins479
- References483
- Chapter 21: Recent Advances on In Vivo Imaging with Fluorescent Proteins485
- I. Macroimaging with Fluorescent Proteins486
- II. Single-Cell In Vivo Imaging with Fluorescent Proteins487
- III. Imaging Dual-Color Angiogenesis and Tumors with Fluorescent Properties488
- IV. Imaging Tumor-Host Interaction with Fluorescent Proteins489
- V. New Applications for Fluorescent Proteins In Vivo: The Development of Effective Bacterial Therapy491
- VI. Conclusions492
- References493
- Chapter 22: Computational Processing and Analysis of Dynamic Fluorescence Image Data497
- I. Introduction498
- II. Rationale499
- A. Why Use Computational Techniques for Quantitative Testing of Biological Hypotheses?500
- B. Role of User Input in Computational Processing and Analysis of Image Data501
- III. Image Features and Representation of Dynamic Events502
- A. Overview502
- B. Image Features502
- C. Representation of Dynamic Events504
- IV. Methods505
- A. Definition of Informative Image Measurements505
- B. Acquisition of Optimized Fluorescent Images506
- C. Filtering of Images509
- D. Detection of Image Features510
- E. Tracking of Features513
- F. Data Analysis516
- V. Two Case Studies of Image Analysis Applied to Mechanistic Cell Biology520
- A. Measurement of Kinetochore MT Length Dynamics in Budding Yeast to Study the Function of Kinetocho520
- B. Tracking of Fluorescent Speckles for the Study of Mechanisms Driving MT Flux in Metaphase Spindle525
- VI. Performance Evaluation for Quality Control527
- A. Algorithms and Software527
- B. Verification of Correctness528
- C. Efficiency Evaluation531
- VII. Summary532
- Acknowledgments533
- References533
- Chapter 23: Automated Classification of Mitotic Phenotypes of Human Cells Using Fluorescent Proteins539
- I. Introduction540
- II. Segmentation of Multicell Images541
- III. Extraction of Image Features544
- IV. Image Features545
- A. Object- and Edge-Related Features545
- B. Haralick Texture Features545
- C. Granularity Features546
- D. Tree-Structured Wavelet Features546
- E. Gray Scale Invariants547
- F. Zernike Moments547
- V. Classification of Mitotic Patterns547
- VI. Experimental Results548
- A. Image Data548
- B. Classification Results550
- VIII. Conclusion552
- Acknowledgments553
- References553
- Chapter 24: Open Tools for Storage and Management of Quantitative Image Data555
- I. Introduction556
- II. Secure, Archived and Available Storage for Biological Image Data557
- III. The Open Microscopy Environment: Data Management Tools for Biological Research559
- A. The OME Server560
- IV. The OMERO Server: A New Server Application for Data Management561
- A. Rationale for a New Server561
- B. Design Criteria for OMERO562
- C. Technical Details of the OMERO Server564
- D. A Flexible Server for Binary Data Applications567
- E. Clients for the OMERO Server: Leading by Design567
- F. Current Capabilities569
- V. Future Directions569
- Acknowledgments570
- References570
- Index571
- Volumes in Series585
This new edition of Fluorescent Proteins presents current applications of autofluorescent proteins in cell and molecular biology authored by researchers from many of the key laboratories in the field. Starting from a current review of the broad palette of fluorescent proteins available, several chapters focus on key autofluorescent protein variants, including spectral variants, photodynamic variants as well as chimeric FP approaches. Molecular applications are addressed in chapters that detail work with single molecules, approaches to generating protein fusions and biosensors as well as analysis of protein-protein interactions in vivo by FRET, fluorescence polarization and fluorescence cross correlation techniques. A number of approaches to in vivo dynamics are presented, including FRAP, photoactivation, and 4-dimensional microscopy. Behavior of spindle components, membrane proteins, mRNA trafficking as well as analysis of cell types in tissues and in development are detailed and provide models for a wide variety of experimental approaches. In addition, several chapters deal directly with the computational issues involved in processing multidimensional image data and using fluorescent imaging to probe cellular behavior with quantitative modeling. This volume brings together the latest perspective and techniques on fluorescent proteins and will be an invaluable reference in a wide range of laboratories.