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GFP: Lighting up Life

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GFP: Lighting up Life PERSPECTIVE GFP: Lighting up life Martin Chalfie1 Department of Biological Sciences, Columbia University, New York, NY 10027 You can observe a lot by watching. Zernike, physics, 1953), large-array ra- My colleagues and I often call their Yogi Berra dio telescopes (Martin Ryle, physics, Nobel Prize the first worm prize. The 1974), the electron microscope (Ernst second went in 2006 to Andy Fire and My companions and I then witnessed Ruska, physics, 1986), the scanning tun- Craig Mello for their discovery of RNA a curious spectacle...TheNautilus neling microscope (Gerd Binnig and interference. I consider this year’s prize floated in the midst of ...trulyliv- Heinrich Rohrer, physics, 1986), com- to be the third worm prize, because if I ing light[,]...aninfinite agglomera- puter-assisted tomography (Allan M. had not worked on C. elegans and con- tion of colored...globules of diaph- Cormack and Godfrey N. Hounsfield, stantly told people that one of its advan- anous jelly.... physiology or medicine, 1979), and, tages was that it was transparent, I am Jules Verne, Twenty Thousand most recently, magnetic resonance imag- convinced I would have ignored GFP Leagues Under the Sea ing (Paul C. Lauterbur and Sir Peter when I first heard of it. These three Now it is such a bizarrely improbable Mansfield, physiology or medicine, prizes speak to the genius of Sydney coincidence that anything so mind- 2003). Brenner in choosing and developing a bogglingly useful could have evolved My road to imaging was not direct. I new organism for biological research. purely by chance that some thinkers had been interested in science from The year before I learned about GFP, have chosen to see it as a final and when I was very young, but after a di- my lab had begun looking at gene expres- clinching proof of the nonexistence sastrous summer lab experience in which sion in the C. elegans nervous system. We of God. every experiment I tried failed, I de- were studying the differentiation and Douglas Adams, The Hitchhiker’s cided on graduating from college that I function of nerve cells needed for mech- Guide to the Galaxy was not cut out to be a scientist. Instead anosensation. Mechanosensors respond to I did a series of somewhat random jobs physical perturbation; they underlie many want to thank the Royal Swedish including teaching high school chemis- of our senses, including touch, hearing, Academy of Sciences and the No- try. During the summer break from and balance. These senses are poorly un- bel Foundation for this amazing teaching, I tried laboratory research one derstood; in particular the transduction and surprising honor. At first I more time, working with Jose´Zadun- molecules, the molecules that detect the Iwondered why I, a biologist and a per- aisky at Yale Medical School (New Ha- mechanical signal, are virtually unknown. son with less than enviable college grades in chemistry, had been selected. ven, CT) (Fig. 1). The successful experi- The genetic studies that I had done with Then I realized that this prize had actu- ments of that summer and his support John Sulston were directed, in part, at ally been given to the GFP molecule, gave me confidence to apply to graduate discovering such transduction molecules. and I am one of its assistants. Thank school, and I entered the physiology de- We thought that by obtaining mutants you for letting me be part of the cele- partment at Harvard (Cambridge, MA) that were defective in touch, which was bration of a wonderful tool for visualiz- in 1972 where I did my thesis with Bob sensed by six cells in the animal, we could ing life. Perlman. Bob and I had a wonderful identify genes that were needed both for Scientific inquiry starts with observa- relationship, which continues to this day. the production and differentiation of tion. The more one can see, the more He is one of the warmest, kindest, and these particular cells and for transduction. one can investigate. Indeed, we often smartest people I know and a great per- In the late 1980s my lab began cloning mark our progress in science by im- son to talk over ideas with. several touch sensitivity genes and testing provements in imaging. The first Nobel My current studies, however, started whether they were expressed in the ani- Prize, the physics prize of 1901, was an when I was accepted as a postdoctoral mal’s touch receptor neurons. At that imaging prize, given to Wilhelm Ro¨nt- fellow by Sydney Brenner at the Medical time three general methods were used to gen for his discovery of X-rays and their Research Council Laboratory of Molec- look at gene and protein expression. The astonishing ability to allow the noninva- ular Biology (Cambridge, U.K.) and be- first was the use of labeled antibodies, sive viewing of the human skeleton. A gan working on the nematode Caeno- whose specificity created outstanding few years later the Nobel Prize in physi- rhabditis elegans. In 2002 Sydney, Bob protein-specific markers. The second was ology or medicine was awarded for the Horvitz, and John Sulston won the No- the use of ␤-galactosidase from the Esche- development of silver nitrate staining to bel Prize in physiology or medicine for richia coli lacZ gene, which could be ex- visualize nerve cells by Camillo Golgi their work on C. elegans. All three pressed as transcriptional and translation and its improvement and use by San- shaped the direction of my research. fusions and visualized by the cleavage and tiago Ramo´n y Cajal to demonstrate the Sydney gave me the opportunity to work subsequent oxidation of X-gal to an insol- cellular nature of the nervous system. with him and an amazingly gifted group This research laid the groundwork of of scientists, Bob, a friend since high modern neurobiology. school, gave me several crucial pieces of Author contributions: M.C. wrote the paper. Over the years several other imaging advice, collaborated on several projects, 1E-mail: [email protected]. techniques and their developers have and served as an example of what one The Nobel Foundation has graciously granted permission to been honored by the Nobel Foundation can achieve in science (I am still follow- print this article: ©The Nobel Foundation 2008. It is a writ- ten version of the Nobel Lecture by Martin Chalfie, ‘‘GFP: for X-ray crystallography (William and ing in his footsteps), and John, with Lighting Up Life.’’ The 2008 Nobel Prize in Chemistry was Lawrence Bragg, physics, 1915; the ul- whom I collaborated the most and who awarded to Dr. Chalfie for demonstrating ‘‘the value of GFP tramicroscope (Richard Zsigmondy, taught me most about how to act honor- as a luminous genetic tag for various biological phenom- chemistry, 1925), NMR (Felix Bloch and ably as a scientist, started me on the ena.’’ From his pioneering work, this protein has become among the most important imaging tools in the life sci- Edward M. Purcell, physics, 1952), the project that still occupies most of my ences, making previously hidden biological processes visi- phase-contrast microscope (Frits time: the study of mechanosensation. ble for study. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0904061106 PNAS ͉ June 23, 2009 ͉ vol. 106 ͉ no. 25 ͉ 10073–10080 Downloaded by guest on September 28, 2021 A B C D Fig. 2. Gene expression methods used before (and after) GFP. (A) Positions of the six touch re- ceptor neurons in C. elegans.(B) Antibody staining to the MEC-7 ␤-tubulin. [Reprinted with permission from ref. 13 (Copyright 1994, Company of Biolo- gists, Ltd.)] (C) ␤-Galactosidase expression of a tran- scriptional fusion for mec-9. (Reprinted from ref. 14 with permission from Elsevier Limited.) (D)In situ hybridization to mec-7 mRNA (image by Sho- hei Mitani). wonderful conversation, found we had similar ideas about what to do with GFP, and decided to collaborate, as Fig. 1. Influences on my career. (Top) The success I had working for Jose´Zadunaisky (Left) convinced me soon as Douglas had finished cloning that maybe I could be a scientist. Bob Perlman (Right) was an outstanding Ph.D. advisor who always had the gene. time to listen to my (often crazy) ideas. (Middle) Working with Sydney Brenner (Left), John Sulston (Right), During that day I learned that GFP and Bob Horvitz (Center) during my postdoctoral years started me on my continued research with C. elegans (Bottom). I am convinced that working with this transparent animal was a major reason I was had several features that made it a very excited about the possibilities of GFP as a biological marker. [Photo credits: photo of Jose´Zadunaisky attractive candidate for a biological (Journal of Experimental Zoology, Vol. 305, No. 1, 2006) reprinted permission of John Wiley & Sons, Inc.); marker: (i) It was a relatively small pro- photo of Bob Perlman (M. Chalfie); photos of Sydney Brenner, John Sulston, and Bob Horvitz (the Nobel tein of only 238 amino acids. (ii)Itwas Foundation); and photo of C. elegans (Adam Antebi).] active as a monomer. (iii) It could be excited by UV or blue light. (iv)Itwasa stable protein that had high quantum uble blue product. The third was in situ work that I subsequently learned had efficiency and did not photobleach eas- hybridization to mRNA. We used all been begun by my colaureate Osamu ily. (v) The active protein did not need a three methods to monitor gene expression Shimomura (1, 2) and then by Jim cofactor or other small molecule to (Fig. 2). Morin and Woody Hastings (3, 4). fluoresce. All three methods had considerable Brehm first spoke about aequorin, which GFP had one feature, however, that limitations; they required extensive and I had heard of as a calcium indicator.
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