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GFP: Lighting up Life GFP: Lighting Up Life You can observe a lot by watching. Yogi Berra My companions and I then witnessed a curious spectacle. .The Nautilus floated in the midst of. truly living light. an infinite agglomeration of colored. globules of diaphanous jelly. Twenty Thousand Leagues Under the Sea – Jules Verne Now it is such a bizarrely improbable coincidence that anything so mind- bogglingly useful could have evolved purely by chance that some thinkers have chosen to see it as a final and clinching proof of the nonexistence of God. The Hitchhiker’s Guide to the Galaxy – Douglas Adams Wilhelm Röntgen Camillo Golgi Santiago Ramón y Cajal Large- Phase Contrast X-ray Crystallography Ultramicroscope Nuclear Magnetic Resonance Array Microscope Radio Telescopes William Lawrence Richard Felix E. M. Frits Martin Bragg Bragg Zsigmondy Bloch Purcell Zernike Ryle Physics, 1915 Chemistry, 1925 Physics, 1952 Physics, 1953 Physics, 1974 Electron Scanning Tunneling Microscope Computer Assisted Tomography Magnetic Resonance Imaging Microscope Ernst Gerd Heinrich Allan Godfrey Paul Peter Ruska Binnig Rohrer Cormack Hounsfield Lauterbur Mansfield Physics, 1986 Physics, 1986 Physiology or Medicine, 1979 Physiology or Medicine, 2003 José Zadunaisky Bob Perlman Sydney Brenner Sydney Brenner Bob Horvitz John Sulston Caenorhabditis elegans Antibody Staining MEC-7 β-galactosidase Activity mec-9 Hongping Du Paul Brehm Aequorea victoria Osamu Shimomura Aequorin + Ca++ light Aequorin + Ca++ + GFP light Douglas Prasher The GFP Fluorophore Tyr66 . NH- Ser65 Phe64 λGFP10 1 TACACACGAA TAAAAGATAA CAAAGATGAG TAAAGGAGAA GAACTTTTCA CTGGAGTTGT 61 CCCAATTCTT GTTGAATTAG ATGGTGATGT TAATGGGCAC AAATTTTCTG TCAGTGGAGA 121 GGGTGAAGGT GATGCAACAT ACGGAAAACT TACCCTTAAA TTTATTTGCA CTACTGGAAA 181 ACTACCTGTT CCATGGCCAA CACTTGTCAC TACTTTCTCT TATGGTGTTC AATGCTTTTC 241 AAGATACCCA GATCATATGA AACAGCATGA CTTTTTCAAG AGTGCCATGC CCGAAGGTTA 361 CAAGTTTGAA GGTGATACCC TTGTTAATAG AATCGAGTTA AAAGGTATTG ATTTTAAAGA 421 AGATGGAAAC ATTCTTGGAC ACAAATTGGA ATACAACTAT AACTCACACA ATGTATACAT 481 CATGGCAGAC AAACAAAAGA ATGGAATCAA AGTTAACTTC AAAATTAGAC ACAACATTGA 541 AGATGGAAGC GTTCAACTAG CAGACCATTA TCAACAAAAT ACTCCAATTG GCGATGGCCC 601 TGTCCTTTTA CCAGACAACC ATTACCTGTC CACACAATCT GCCCTTTCGA AAGATCCCAA 661 CGAAAAGAGA GACCACATGG TCCTTCTTGA GTTTGTAACA GCTGCTGGGA TTACACATGG 721 CATGGATGAA CTATACAAAT AAATGTCCAG ACTTCCAATT GACACTAAAG TGTCCGAACA Eco781RI ATTACTAAAA TCTCAGGGTT CCTGGTTAAA TTCAGGCTGA GATATTATTT EcoRI ATATATTTAT 841 AGATTCATTA AAATTGTATG AATAATTTAT TGATGTTATT GATAGAGGTT ATTTTCTTAT Ghia Euskirchen Yuan Tu Bill Ward Green Fluorescent Protein: A New Marker for Gene Expression The Aequorea victoria Green Fluorescent Protein Needs No Exogenously-Added Component to Produce a Fluorescent Product in Prokaryotic and Eukaryotic Cells Green Fluorescent Protein as a Marker for Gene Expression Martin Chalfie, Yuan Tu, Ghia Euskirchen, William W. Ward, Douglas C. Prasher Science 263: 802-805, 1994 Tulle Hazelrigg Sarah Chalfie Implications for bcd mRNA localization from spatial distribution of exu protein in Drosophila oogenesis Shengxian Wang and Tulle Hazelrigg Nature 369: 400-403, 1994 Tulle Hazelrigg Shengxian Wang Advantages of GFP as a Biological Marker 1. Heritable 2. Relatively Non-invasive 3. Small and Monomeric 4. Visible in Living Tissues Improving GFP Tyr66 Emission Ser65 Excitation Roger Tsien Papers Using Green Fluorescent Protein The First Human GFP Transgenic? Ang Lee Gene Expression Pmec-17 gfp ALM PVM PLM AVM Yun Zhang Co-expression EGL-44::YFP EGL-46::CFP MERGED FLP FLP FLP Ji Wu Protein Localization MEC-1 MEC-9 MEC-5 Kunitz/EGF proteinsPmec-4collagencfp MEC-4 MEC-10 DEG/ENaC MEC-6 paraoxonase MEC-2 UNC-24 PHB-domain proteins MEC-7 β-tubulin MEC-12 α-tubulin mec-4::yfp Dattananda Chelur Mutant Screens and Characterizstion unc-51 Wild Type mec-7 Hongping Du Visualizing Synapses Mike Nonet Pmec-7 rab-3 gfp wild type mec-15 Alex Bounoutas Cell Isolation Irini Topalidou FLP neurons Touch neurons Yun Zhang The Problem with C. elegans Electrophysiology Cell-specific Electrophysiology MEC-4 MEC-10 MEC-6 MEC-2 Bob O’Hagan Miriam Goodman Non-covalent Reconstitution of GFP Interaction of NZ CZ Leucine Zippers linker linker NGFP CGFP NZGFP CZGFP Reconstitution of Functional GFP From Promoter A From Promoter B recGFP A B Indraneel Ghosh, Andrew D. Hamilton, and Lynne Regan (2000) Antiparallel Leucine Zipper-Directed Protein Reassembly: Lynne Regan Application to the Green Fluorescent Protein. J. Am. Chem. Soc. 122: 5658–5659. Refining Cell Labeling ALM Intestine AVM HSN PVM FLP Other PVD neurons PLM Hypodermis mec-3 egl-44 Pmec-3 nzgfp & Pegl-44 czgfp Shifang Zhang Chuck Ma Pmec-18 praja gfp L1 L4 Adult Control Chuck Ma Colchicine Mutagenesis Colchicine Leslie Emtage MEC-3 MEC-7 + MEC-12 microtubules Homeodomain β-tubulin α-tubulin Transcription Factor Alex Bounoutas Madeline Friedlen Vivian Chalfie Eli Chalfie Postdoctoral Bill Walthall John Kratz Angela Molly Weiner Scientists Jeff Way Joy Liang Georgopoulos Chris William Eve Wolinsky Shujun Luo Rachel Goldstein Anahita Amiri Cathy Savage Judith Green Technicians Guy Caldwell Graduate Jim Taub Peter Homer Lucinda Carnell Students Ji Wu Rafaz Hoque Margaret Barnes Anne Chambers Yi-Chun Wu James Hudspeth Elke Bergholz Dattananda Alex Bounoutas Ding Xue Shari Jawetz Lei Chen Chelur Andrea Calixto Shifang Zhang Paul Josephson Ellen Dean Brian Coblitz Xiaoyin Chen Yun Zhang Eric Kanter Nora Hom Monica Driscoll Yushu Chen Lexy Kovach Maud Kinnell Chip Ferguson Hongping Du Undergraduates Joe Lau Connie Mauoka Nikolaos George Anne Duggan Sam Lee Evelyn Reilly Miriam Goodman Lesley Emtage Macy Au Lorraine Yuan Tu Jang Hee Hahn Glen Ernstrom Paul Auwaeter Lothringer Yingzi Xue Chris Li Ghia Euskirchen Peggy Brickman David Meshoulam Andrea Yao Charles Ma Jaime García- Kate Brauman Jeremy Mindich Chris Martin Añoveros John Byun Mariya Rosenblit Faculty Shohei Mitani Guoqiang Gu Alex Chang Julie Rosenthal Visitors Erich Schwarz Mingxia Huang Victor Chang Aaron Scheffler Anne Toker Siavash Iris Chin Shai Shaham Christian Neri Irini Topalidou Karimzadegan Bradley Collins Jay Srinivasan Jonathan Millet Treinin Charles Keller Michael Dreyfuss Leslie Vosshall Rothblatt .
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    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.
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