GFP: an illuminating tool: Green Fluorescent Proteins (Methods in Cell Biology, Vol. 58), edited by Kevin F. Sullivan and Steve A. Kay

Abstract

Academic Press, 1999. $64.95 (386 pages)ISBN 0 12 676075 6Within the past four years, variants of the green-fluorescent protein (GFP) have become standard tools in cell biology. When Prasher and colleagues first cloned the gene for GFP from the jellyfish Aequorea victoria in 19921xPrasher, D.C. et al. Gene. 1992; 111: 229–233Crossref | PubMed | Scopus (1425)See all References1 it was not obvious that GFP would become so useful that it would warrant its own volume of Methods in Cell Biology. Initial problems with dim fluorescence, poor expression and lack of spectral variants have now been solved, making GFP an effective fluorescent tag for most cloned proteins. GFP is remarkably inert; fusions to GFP are quite often functional or at least localize properly despite an additional 237 amino acids of polypeptide sequence. GFP is also remarkably resistant to photobleaching, allowing long-term time-lapse microscopy studies. This book covers most of the broad range of applications cell biologists have found for GFP, with chapters from many of the labs that were involved in overcoming the initial problems. It is a comprehensive guide to GFP, useful both for someone starting to work with GFP and for those with considerable experience with GFP who want to see how others are using it.The style is consistent with the traditional format of Methods in Cell Biology; the book is organized by chapters on a particular topic, some quite specific, each with detailed protocols, descriptions of techniques and advice from the authors – all the helpful and sometimes critical details that are never published in research papers owing to space constraints. At the end of each chapter are extensive references that make a useful guide for further reading. The disadvantage of this format is that Green Fluorescent Proteins is far from concise. There is considerable overlap because each chapter was written separately – often on topics that use similar techniques.The range of topics is broad, beginning with three chapters that cover the biophysics and structural basis of GFP fluorescence and quantitative imaging with GFP, which provide a basic foundation for the more applied chapters that comprise the rest of the book. The majority of these chapters describe strategies for generating GFP-fusions and techniques for time-lapse imaging a variety of biological systems, from yeast to flies to tissue-culture cells. Other chapters cover single-molecule experiments with GFP, fluorescence resonance energy transfer (FRET), photobleaching techniques, multicolour imaging with GFP variants and cell sorting based on GFP fluorescence. The final chapter discusses practical considerations for setting up an imaging facility – computers, backup systems, image processing, space and ventilation.In general, Green Fluorescent Proteins is quite comprehensive, but there were several additional things that would have been useful to include. Two techniques could have been described more completely. The first is a method to tag proteins indirectly with GFP based on noncovalent heterodimerization of GFP and cytoplasmic structural proteins2xKatz, B.Z. et al. Biotechniques. 1998; 25: 298–302304PubMedSee all References2. Dimerization is mediated by a modified leucine-zipper protein spacer. This method seems to allow tagging of structural proteins whose assembly might be hindered by direct fusion to GFP. The second is a complete description of the recently characterized two-colour double-labelling technique based on sequential excitation of the cyan (optimized W7) and yellow (optimized 10C) spectral variants of GFP3xSee all References3. Spectral variants and double labelling are discussed briefly and rather speculatively in several chapters, but a thorough characterization of an effective technique is required. The method using cyan and yellow variants is the only technique that is amenable to long-term time-lapse imaging and photobleaching studies. In addition, movies of protein dynamics visualized with GFP are often quite dramatic and always informative, so I would have liked to have seen an accompanying CD-ROM with movies and images (such as Ref. 4xSee all ReferencesRef. 4). Finally, although some might feel it outside the scope of the book, a chapter on alternatives to GFP and future improvements in visualizing specific proteins in live cells using fluorescent probes would have provided valuable perspective and interesting directions for future experiments.Green Fluorescent Proteins fills an important niche as a detailed guide to using GFP variants to solve a variety of problems in cell biology. When people come to me with questions about GFP, I now refer them to this book.