Methods in Cell Biology Vol. 58 : Green Fluorescent Proteins

by Wilson, Leslie; Matsudaira, Paul

• ISBN13: 9780125441605
• ISBN10: 0125441606
• Format: Hardcover
• Copyright: 1998-11-01
• Publisher: Elsevier Science Serials

Summary

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

Table of Contents

Contributors	xi	(4)
Preface	xv
1. Biophysics of the Green Fluorescent Protein	1	(18)
F.G. Prendergast
I. Introduction	1	(2)
II. Protein Folding and the Generation of This Chromophore	3	(5)
III. The Biophysics of the Fluorescence of GTP	8	(7)
IV. Resonance Energy Transfer Involving GFP	15	(1)
V. Summary	16	(1)
References	17	(2)
2. Understanding Structure-Function Relationships in the Aequorea victoria Green Fluorescent Protein	19	(12)
Andrew B. Cubitt
Leslie A. Woollenweber
Roger Heim
I. Introduction	19	(1)
II. Structure	20	(1)
III. Chromophore Formation	21	(1)
IV. Effects of Mutations on the Spectroscopic Properties of GFP	22	(4)
V. Effects of Mutations That Improve Thermosensitivity	26	(1)
VI. The Development of Enhanced Mutants	27	(2)
References	29	(2)
3. Quantitative Imaging of the Green Fluorescent Protein (GFP)	31	(18)
David W. Piston
George H. Patterson
Susan M. Knobel
I. Introduction	31	(1)
II. Factors That Influence/Limit Quantitation of GFP in Fluorescence Microscopy	32	(10)
III. Applications of LSCM for Quantitative Imaging of GFP	42	(4)
IV. Preparation of Purified GFP Samples	46	(1)
References	47	(2)
4. Single-Molecule Fluorescence Detection of Green Fluorescence Protein and Application to Single-Protein Dynamics	49	(26)
Daniel W. Pierce
Ronald D. Vale
I. Introduction	49	(2)
II. Design Considerations for Fluorescence Microscopes for Single-Molecule Detection	51	(4)
III. Characteristics of the Fluorescence from Single GFP Molecules	55	(9)
IV. Advantages of Using GFP for Single-Molecule Detection	64	(1)
V. GFP in Vitro and in Vivo Assays	65	(2)
Appendix I. Details of the TIR Microscope	67	(4)
Appendix II. Data Acquisition and Analysis	71	(1)
References	72	(3)
5. Targeting GFP to Organelles	75	(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	75	(1)
II. Construction and Expression of the Organelle-Targeted GFP Chimeras	76	(3)
III. Dynamic Monitoring of Organelle Structure with the Targeted GFPs	79	(1)
IV. Expression in Primary Cultures	80	(1)
V. Visualizing GFP Chimeras with Different Spectral Properties	81	(1)
VI. Protocols	82	(3)
References	85	(2)
6. Cytoskeletal Dynamics in Yeast	87	(20)
Janet L. Carminati
Tim Stearns
I. Introduction	87	(1)
II. Generating GFP Fusions	88	(5)
III. Imaging Considerations for Yeast Cells	93	(1)
IV. Time-Lapse Microscopy	94	(2)
V. Results: Cytoskeletal GFP Fusion Proteins	96	(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