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