Methods in Cell Biology Vol. 58 : Green Fluorescent Proteins

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ISBN: 9780125441605 | 0125441606
Cover: Hardcover
Copyright: 11/1/1998

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This volume is an authoritative and comprehensive treatment of the approaches and techniques used for Green Fluorescent Proteins (GFP). The primary focus of this work is on research using biological systems. The volume covers all aspects of GFP, from its expression in different organisms to specific microscopic and data analysis methods. Key Features * Only volume on Green Fluorescent Protein research * Covers all aspects of GFP * Provides specific microscopic and data analysis methods * Discusses the design and construction of GFP fusion proteins * Covers GFP expression in animals, insects, plants, and microbes * Details procedures for time lapse imaging of living cells * Explains how to implement single molecule fluorescence detection with GFP * Discusses dual label GFP strategies for multicolor fluorescence * Presents fluorescence resonance energy transfer methods with GFPs * Details quantitative fluorescence imaging techniques * Extensively illustrated with color photographs

Contributors

(4)

Preface

1. Biophysics of the Green Fluorescent Protein

(18)

F.G. Prendergast

I. Introduction

(2)

II. Protein Folding and the Generation of This Chromophore

(5)

III. The Biophysics of the Fluorescence of GTP

(7)

IV. Resonance Energy Transfer Involving GFP

(1)

V. Summary

(1)

References

(2)

2. Understanding Structure-Function Relationships in the Aequorea victoria Green Fluorescent Protein

(12)

Andrew B. Cubitt

Leslie A. Woollenweber

Roger Heim

I. Introduction

(1)

II. Structure

(1)

III. Chromophore Formation

(1)

IV. Effects of Mutations on the Spectroscopic Properties of GFP

(4)

V. Effects of Mutations That Improve Thermosensitivity

(1)

VI. The Development of Enhanced Mutants

(2)

References

(2)

3. Quantitative Imaging of the Green Fluorescent Protein (GFP)

(18)

David W. Piston

George H. Patterson

Susan M. Knobel

I. Introduction

(1)

II. Factors That Influence/Limit Quantitation of GFP in Fluorescence Microscopy

(10)

III. Applications of LSCM for Quantitative Imaging of GFP

(4)

IV. Preparation of Purified GFP Samples

(1)

References

(2)

4. Single-Molecule Fluorescence Detection of Green Fluorescence Protein and Application to Single-Protein Dynamics

(26)

Daniel W. Pierce

Ronald D. Vale

I. Introduction

(2)

II. Design Considerations for Fluorescence Microscopes for Single-Molecule Detection

(4)

III. Characteristics of the Fluorescence from Single GFP Molecules

(9)

IV. Advantages of Using GFP for Single-Molecule Detection

(1)

V. GFP in Vitro and in Vivo Assays

(2)

Appendix I. Details of the TIR Microscope

(4)

Appendix II. Data Acquisition and Analysis

(1)

References

(3)

5. Targeting GFP to Organelles

(12)

Francesca De Giorgi

Zimran Ahmed

Carlo Bastianutto

Marisa Brini

Laurence Sophie Jouaville

Robert Marsault

Marta Murgia

Paolo Pinton

Tullio Pozzan

Rosario Rizzuto

I. Introduction

(1)

II. Construction and Expression of the Organelle-Targeted GFP Chimeras

(3)

III. Dynamic Monitoring of Organelle Structure with the Targeted GFPs

(1)

IV. Expression in Primary Cultures

(1)

V. Visualizing GFP Chimeras with Different Spectral Properties

(1)

VI. Protocols

(3)

References

(2)

6. Cytoskeletal Dynamics in Yeast

(20)

Janet L. Carminati

Tim Stearns

I. Introduction

(1)

II. Generating GFP Fusions

(5)

III. Imaging Considerations for Yeast Cells

(1)

IV. Time-Lapse Microscopy

(2)

V. Results: Cytoskeletal GFP Fusion Proteins

(7)

VI. Future

103

(1)

References

104

(3)

7. Analysis of Nuclear Transport in Vivo

107

(16)

Paul Ferrigno

Pamela A. Silver

I. Introduction

107

(1)

II. Experimental Approaches and Protocols

108

(13)

References

121

(2)

8. GFP Fusion Proteins as Probes for Cytology in Fission Yeast

123

(16)

Kenneth E. Sawin

I. Introduction

123

(1)

II. Expressing GFP Fusion Proteins

124

(7)

III. Applications of Fusion Proteins

131

(6)

References

137

(2)

9. GFP Variants for Multispectral Imaging of Living Cells

139

(14)

James Haseloff

I. Introduction

139

(1)

II. Green Fluorescent Protein Markers

140

(3)

III. Imaging of Living Cells

143

(3)

IV. Marking Different Cell Types in Arabidopsis

146

(1)

V. Spectrally Distinct Fluorescent Proteins for Multichannel Confocal Microscopy

147

(2)

VI. Summary

149

(1)

References

150

(3)

10. GFP Fusions to a Microtubule Motor Protein to Visualize Meiotic and Mitotic Spindle Dynamics in Drosophila

153

(12)

Sharyn A. Endow

I. Introduction

153

(1)

II. Labeling Strategies

154

(2)

III. Imaging GFP

156

(3)

IV. Applications of Ncd-GFP Imaging

159

(3)

V. Perspectives

162

(1)

References

162

(3)

11. GFP as a Cell and Developmental Marker in the Drosophila Nervous System

165

(18)

Andrea Brand

I. Introduction

165

(3)

II. Targeted Expression of GFP in Drosophila

168

(1)

III. Lines for Expression of GFP

168

(7)

IV. Visualizing GFP Expression

175

(5)

References

180

(3)

12. Using Time-Lapse Confocal Microscopy for Analysis of Centromere Dynamics in Human Cells

183

(20)

Kevin F. Sullivan

Richard D. Shelby

I. Introduction

183

(1)

II. GFP Fusion Proteins

184

(1)

III. Microscopy

185

(5)

IV. Analysis

190

(4)

V. Summary

194

(1)

Appendix: Handling Confocal Images on the Laboratory Computer

195

(5)

References

200

(3)

13. Visualization of Large-Scale Chromatin Structure and Dynamics Using the lac Operator/lac Repressor Reporter System

203

(21)

Andrew S. Belmont

Gang Li

Gail Sudlow

Carmen Robinett

I. Introduction

203

(1)

II. Overview of Methodology

204

(2)

III. Construction of the lac Operator Repeat

206

(2)

IV. Manipulation of the lac Operator Repeats

208

(2)

V. Repressor-NLS and GFP-Repressor-NLS Constructs

210

(2)

VI. Gene Amplification and Cell Cloning

212

(2)

VII. Repressor Staining and Immunodetection of the lac Operator Repeat

214

(1)

VIII. In Vivo Observation of GFP-Repressor Localization

215

(1)

IX. Phototoxicity Issues

216

(2)

X. Present Results and Future Directions

218

(2)

References

220

(4)

14. Centrosome Dynamics in Living Cells

224

(16)

Aaron Young

Richard Tuft

Walter Carrington

Stephen J. Doxsey

I. Introduction

224

(1)

II. Cloning and Expression of GFP-Pericentrin

225

(3)

III. High-Speed Microscopy

228

(3)

IV. Image Restoration by an Improved Deconvolution Method

231

(3)

V. Imaging Centrosomes

234

(1)

VI. Postimaging Confirmation of Centrosome Integrity and Function

235

(3)

References

238

(2)

15. Transfections of Primary Muscle Cell Cultures with Plasmids Coding for GFP Linked to Full-Length and Truncated Muscle Proteins

240

(21)

Guissou A. Dabiri

Kenan K. Turnacioglu

Joseph C. Ayoob

Jean M. Sanger

Joseph W. Sanger

I. Introduction

240

(1)

II. Construction of GFP-Linked Muscle Proteins

241

(2)

III. Preparation of Embryonic Avian Cardiomyocytes and Skeletal Muscle Myoblasts

243

(1)

IV. Methods of Transfection of Cross-Striated Cells in Culture

244

(3)

V. Transfection of Cross-Striated Muscle Cells with Full-Length cDNA for Sarcomeric Proteins

247

(8)

VI. Microscopic Observations of Live Cells

255

(2)

VII. Postprocessing of Transfected Cells

257

(1)

VIII. Problems Encountered in Cells Transfected with GFP-Sarcomeric Proteins

257

(1)

IX. Overview

258

(1)

References

258

(3)

16. Monitoring the Dynamics and Mobility of Membrane Proteins Tagged with Green Fluorescent Protein

261

(22)

J. Lippincott-Schwartz

J.F. Presley

K.J.M. Zaal

K. Hirschberg

C.D. Miller

J. Ellenberg

I. Introduction

261

(1)

II. Constructing and Expressing GFP Fusion Proteins: Strategies for Optimizing Brightness and Assessing Chimera Function

262

(2)

III. Practical Guidelines for the Preparation and Imaging of GFP-Expressing Cells

264

(1)

IV. Time-Lapse Imaging of GFP Chimeras: Critical Parameters

265

(3)

V. Analysis of Time-Lapse Imaging Data

268

(2)

VI. Relating GFP Chimera Fluorescence to Actual Numbers of GFP Molecules

270

(1)

VII. Fluorescence Recovery after Photobleaching FRAP

271

(1)

VIII. Qualitative FRAP Experiments

272

(3)

IX. Quantitative FRAP

275

(1)

X. Calculating D

276

(2)

XI. Fluorescence Loss in Photobleaching (FLIP) Using a Confocal Microscope

278

(2)

XII. Other Applications of Photobleaching

280

(1)

References

280

(3)

17. Synchronous Real-Time Reporting of Multiple Cellular Events

283

(11)

Jeffrey D. Plautz

Steve A. Kay

I. Introduction

283

(1)

II. Green Fluorescent Protein

284

(1)

III. Luciferase

285

(1)

IV. Instrumentation and Techniques

286

(4)

V. Future Directions

290

(1)

References

291

(3)

18. Visualizing Protein Interactions in Living Cells Using Digitized GFP Imaging and FRET Microscopy

294

(21)

Ammasi Periasamy

Richard N. Day

I. Introduction

294

(1)

II. The Theory of FRET

295

(2)

III. Review of the FRET Literature

297

(1)

IV. Why Use FRET Microscopy?

298

(1)

V. Why Use the GEPs for FRET?

298

(3)

VI. Some Considerations for the Use of GFPs in FRET Imaging

301

(2)

VII. Some Considerations for Designing a FRET Imaging System

303

(4)

VIII. The Practical Application of FRET to Visualize Protein-Protein Interactions

307

(2)

IX. Overview and Conclusion

309

(1)

References

310

(5)

19. Flow Cytometric Analysis and FACS Sorting of Cells Based on GFP Accumulation

315

(29)

David W. Galbraith

Michael T. Anderson

Leonard A. Herzenberg

I. General Introduction

315

(4)

II. Methods and Specific Applications

319

(9)

III. Typical Results

328

(7)

IV. Discussion and Conclusions

335

(3)

References

338

(6)

20. GFP Biofluorescence: Imaging Gene Expression and Protein Dynamics in Living Cells

344

(25)

Paul C. Goodwin

I. Introduction

344

(1)

II. Facilities

345

(2)

III. Maintaining Cells

347

(2)

IV. Imaging Systems

349

(6)

V. Computer Systems

355

(4)

VI. Output

359

(6)

VII. Conclusions

365

(1)

References

366

(3)

Index

369

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Methods in Cell Biology Vol. 58 : Green Fluorescent Proteins

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