edited by Ed Manser and Thomas Leung Humana Press (2002). 267 pages. ISBN 0-89603-934-X $99.50
Few families of signalling proteins rival the Ras-related small GTPases in breadth of cellular function. Since identification of Ras as a key oncogene in human cancer 20 years ago, well over 100 related mammalian proteins have been recorded, together with a host of relatives in simpler eukaryotes. Cell biologists find these proteins particularly hard to avoid — small GTPases are key regulators of some of the most intensely researched problems in cell biology. The 50 or so human Rab GTPases alone are enough to keep membrane traffickers quiet for the foreseeable future.
In GTPase Protocols, Manser and Leung assemble the key protocols required for researchers venturing into this field, together with inspiration for those of us whose talk of `heroic' pull-down purifications somehow never makes it into the cold room. The publishers state that the book is“written by international experts who, in many cases, have pioneered the technique”, and this is evident from both the impressive list of contributors and the quality and detail of the presented methods.
The book is organised into two parts: a shorter first section describing general protocols, followed by specific methods for each of the five small GTPase subfamilies. In some ways this feels like a slightly artificial division — Zhao and Lim's update of their classic blot overlay protocol is a great general method that deserves to be liberated from the Ras techniques section. Similarly, the excellent description by Takai and co-workers of the isolation of Rab3 effectors by affinity chromatography is one that could be usefully applied to any other small GTPase. These are minor quibbles that in part reflect the commonalities between the small GTPase subfamilies.
For most of us, our initiation into small GTPase work will involve throwing dominant-negative mutants at our system and hoping for the best. Fittingly,the book begins with a thorough introduction to these experimental tools in which Manser explains both the basis of their action and, most importantly,the caveats of their use. This is an example of where the book is at its strongest — the combination of clear practical detail with underlying theory provides the reader with the information required not only to perform the technique but also to interpret the resulting data. Other basic techniques are handled in this section with equal clarity. Smith and Rittinger provide an exhaustive description of the purification of recombinant small GTPases from bacteria. Here, it would have been nice to see some discussion of purification of post-translationally-modified small GTPases from insect cells, although this is dealt with briefly elsewhere. Ahmadian et al. give a precise account of fluorescent techniques for the kinetic analysis of small GTPases, including assays for guaninenucleotide-exchange factors and GTPase-activating proteins. What can seem quite complicated assays are explained in a straightforward manner, with the fringe benefit of reducing unnecessary graduate student irradiation. After three of the `bread and butter' techniques of small GTPase work, the section closes with a general technique for the more adventurous:Van Aelst and colleagues giving a detailed description of cDNA-RDA screens for identifying genes regulated by small GTPases.
The subsequent sections deal with specific protocols for the Ras, Rho, Rab,Arf and Ran GTPases although, as mentioned before, there is plenty of potential for cross-pollination between these subject headings. For me, the standout specific techniques are the protocols for assaying Rho GTPases and for measuring cell migration and invasion, although this is probably coloured in part by my own experimental frustrations. Workers on other small GTPase subfamilies will also find succour: there are detailed protocols for the post-translational modification of Rab and Arf GTPases and an excellent section on Ran GTPases. As with most books of experimental methods, there are occasional chapters that read like lightly revised methods sections from previously published work but these are few and far between.
In summary, this is a collection of detailed and precise experimental methods from some of the leading research groups in this field. The authors are to be congratulated on the clarity brought to each technique, and thanked in advance for dealing with the flood of requests for reagents that their involvement will undoubtedly provoke.