GFP: Lighting Up Life You can observe a lot by watching. Yogi Berra - PowerPoint PPT Presentation

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

Camillo Golgi Santiago Ramón y Cajal Wilhelm Röntgen

Large- Phase Contrast Array X-ray Crystallography Ultramicroscope Nuclear Magnetic Resonance Microscope Radio Telescopes Martin William Lawrence Richard Felix E. M. Frits Ryle Zernike Bragg Bragg Zsigmondy Bloch Purcell Physics, 1974 Physics, 1915 Chemistry, 1925 Physics, 1952 Physics, 1953 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

Aequorea victoria Osamu Shimomura Paul Brehm Aequorin + Ca ++ light Aequorin + Ca ++ + GFP light

Douglas Prasher

The GFP Fluorophore Tyr 66 . . . . . . NH- Ser 65 Phe 64

λ GFP10 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 Eco RI Eco RI 781 ATTACTAAAA TCTCAGGGTT CCTGGTTAAA TTCAGGCTGA GATATTATTT 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 Tyr 66 Emission Ser 65 Excitation Roger Tsien

Papers Using Green Fluorescent Protein

The First Human GFP Transgenic? Ang Lee

Gene Expression P mec-17 gfp PVM ALM PLM AVM Yun Zhang

Co-expression EGL-44::YFP EGL-46::CFP MERGED FLP FLP FLP Ji Wu

Protein Localization MEC-1 MEC-9 P mec-4 cfp MEC-5 Kunitz/EGF proteins collagen DEG/ENaC MEC-10 MEC-4 MEC-6 paraoxonase MEC-2 UNC-24 MEC-7 PHB-domain proteins 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 P mec-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 Reconstitution of NZGFP CZGFP 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 Intestine ALM HSN AVM PVM Other FLP neurons PVD Hypodermis PLM mec-3 egl-44 P mec-3 nzgfp & P egl-44 czgfp Shifang Zhang Chuck Ma

P mec-18 praja gfp L1 L4 Adult Control Chuck Ma Colchicine Mutagenesis Colchicine Leslie Emtage microtubules MEC-3 MEC-7 + MEC-12 Homeodomain β -tubulin α -tubulin Transcription Alex Bounoutas Factor

Madeline Friedlen Vivian Chalfie Eli Chalfie

John Kratz Angela Molly Weiner Postdoctoral Bill Walthall Scientists Jeff Way Joy Liang Georgopoulos Chris William Eve Wolinsky Shujun Luo Rachel Goldstein Anahita Amiri Cathy Savage Judith Green Technicians Jim Taub Peter Homer Guy Caldwell Graduate 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 Shifang Zhang Paul Josephson Ellen Dean Chelur Andrea Calixto 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 Sam Lee Evelyn Reilly Nikolaos George Anne Duggan 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 Jaime García- Kate Brauman Jeremy Mindich Charles Ma 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 Siavash Iris Chin Shai Shaham Christian Neri Anne Toker Irini Topalidou Karimzadegan Bradley Collins Jay Srinivasan Jonathan Millet Treinin Charles Keller Michael Dreyfuss Leslie Vosshall Rothblatt