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

GFP: Lighting Up Life

You can observe a lot by watching.Yogi Berra

Wilhelm Röntgen Santiago Ramón y Cajal Camillo Golgi

Ultramicroscope

RichardZsigmondy

Chemistry, 1925

Phase Contrast Microscope

FritsZernike

Physics, 1953

Large-

ArrayRadio

Telescopes

MartinRyle

Physics, 1974

ElectronMicroscope

ErnstRuska

Physics, 1986

X-ray Crystallography

Physics, 1915

LawrenceBragg

WilliamBragg

Nuclear Magnetic Resonance

Physics, 1952

E. M.Purcell

FelixBloch

Scanning Tunneling Microscope

Physics, 1986

HeinrichRohrer

GerdBinnig

Computer Assisted Tomography

Physiology or Medicine, 1979

Godfrey Hounsfield

AllanCormack

Magnetic Resonance Imaging

Physiology or Medicine, 2003

PeterMansfield

PaulLauterbur

Sydney BrennerJosé

Zadunaisky Bob Perlman

Sydney Brenner Bob Horvitz John Sulston

Caenorhabditis elegans

β-galactosidase Activity

mec-9

Antibody Staining

MEC-7

Hongping Du

Aequorin + Ca++

+ GFP light

Aequorin + Ca++ light

Paul Brehm Osamu ShimomuraAequorea victoria

Douglas Prasher

The GFP Fluorophore

Tyr66

Phe64

Ser65

. . .

. . . NH-

1

TACACACGAA TAAAAGATAA CAAAGATGAG TAAAGGAGAA GAACTTTTCA CTGGAGTTGT61 CCCAATTCTT GTTGAATTAG ATGGTGATGT TAATGGGCAC AAATTTTCTG

TCAGTGGAGA121 GGGTGAAGGT GATGCAACAT ACGGAAAACT TACCCTTAAA TTTATTTGCA CTACTGGAAA181 ACTACCTGTT CCATGGCCAA CACTTGTCAC TACTTTCTCT TATGGTGTTC AATGCTTTTC241 AAGATACCCA GATCATATGA AACAGCATGA CTTTTTCAAG AGTGCCATGC CCGAAGGTTA361 CAAGTTTGAA GGTGATACCC TTGTTAATAG AATCGAGTTA AAAGGTATTG ATTTTAAAGA421 AGATGGAAAC ATTCTTGGAC ACAAATTGGA ATACAACTAT AACTCACACA ATGTATACAT481 CATGGCAGAC AAACAAAAGA ATGGAATCAA AGTTAACTTC AAAATTAGAC ACAACATTGA541 AGATGGAAGC GTTCAACTAG CAGACCATTA TCAACAAAAT ACTCCAATTG GCGATGGCCC601 TGTCCTTTTA CCAGACAACC ATTACCTGTC CACACAATCT GCCCTTTCGA AAGATCCCAA661 CGAAAAGAGA GACCACATGG TCCTTCTTGA GTTTGTAACA GCTGCTGGGA TTACACATGG721 CATGGATGAA CTATACAAAT AAATGTCCAG ACTTCCAATT GACACTAAAG TGTCCGAACA781 ATTACTAAAA TCTCAGGGTT CCTGGTTAAA TTCAGGCTGA GATATTATTT ATATATTTAT841 AGATTCATTA AAATTGTATG AATAATTTAT TGATGTTATT GATAGAGGTT ATTTTCTTAT

λGFP10

EcoRI EcoRI

Ghia

Euskirchen

Yuan Tu

Bill Ward

Green Fluorescent Protein: A New Markerfor Gene Expression

The Aequorea victoria Green Fluorescent Protein Needs No Exogenously-Added Component to Produce a Fluorescent

Product in Prokaryotic and Eukaryotic Cells

Martin Chalfie, Yuan Tu, Ghia

Euskirchen, William W. Ward, Douglas C. Prasher

Science 263: 802-805, 1994

Green Fluorescent Protein as a Markerfor Gene Expression

Tulle Hazelrigg

Sarah Chalfie

Shengxian

Wang and Tulle Hazelrigg

Shengxian

Wang

Implications for bcd mRNA localization from spatial distribution of exu protein in Drosophila oogenesis

Nature 369: 400-403, 1994Tulle Hazelrigg

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 ExcitationRoger Tsien

Papers Using Green Fluorescent Protein

The First Human GFP Transgenic?

Ang

Lee

PVM

AVMPLM

ALMPmec-17 gfp

Gene Expression

Yun

Zhang

FLP FLP FLP

EGL-44::YFP EGL-46::CFP MERGED

Co-expression

Ji

Wu

Pmec-4 cfp

mec-4::yfpDattananda

Chelur

Protein Localization

MEC-1 MEC-9 MEC-5

MEC-7MEC-12

MEC-4 MEC-10

MEC-6

MEC-2β-tubulin

α-tubulin

collagenKunitz/EGF proteins

DEG/ENaC

PHB-domain proteins

paraoxonase

UNC-24

unc-51

mec-7

Wild Type

Mutant Screens and Characterizstion

Hongping

Du

gfprab-3

Mike Nonet

Visualizing Synapses

wild type

mec-15

Alex Bounoutas

Pmec-7

Yun

Zhang

Cell Isolation

Irini

Topalidou

Touch neuronsFLP neurons

The Problem with C. elegans Electrophysiology

Bob O’Hagan

Cell-specific Electrophysiology

MEC-4 MEC-10

MEC-6

MEC-2

Miriam

Goodman

Interaction ofLeucine

Zippers

Reconstitution of Functional GFP

recGFP

NZGFP

NZ CZ

linker

NGFP CGFP

linker

CZGFPFrom Promoter A From Promoter B

A B

Non-covalent Reconstitution of GFP

Lynne Regan

Indraneel

Ghosh, Andrew D. Hamilton, and Lynne Regan (2000) Antiparallel

Leucine

Zipper-Directed Protein Reassembly:

Application to the Green Fluorescent Protein. J. Am. Chem. Soc. 122: 5658–5659.

Refining Cell Labeling

mec-3 egl-44

FLP

Intestine

Pmec-3 nzgfp & Pegl-44 czgfp

PVDPLM

AVMALM

PVM

Hypodermis

HSNOther

neurons

Shifang

Zhang Chuck Ma

L1 L4 Adult

Control

Colchicine

Mutagenesis

Colchicine

Alex Bounoutas

Chuck Ma

Leslie Emtage

praja gfpPmec-18

MEC-3 MEC-7 + MEC-12 microtubulesHomeodomainTranscription

Factor

β-tubulin α-tubulin

Madeline Friedlen

Vivian Chalfie Eli Chalfie

Postdoctoral Scientists

Anahita

AmiriGuy Caldwell Lucinda CarnellAnne ChambersDattananda

ChelurBrian CoblitzMonica Driscoll Chip Ferguson Nikolaos

George Miriam GoodmanJang Hee

HahnChris Li Charles Ma Chris MartinShohei

MitaniErich Schwarz Anne TokerIrini

TopalidouMillet Treinin

Bill Walthall Jeff WayEve Wolinsky

Graduate Students

Alex BounoutasAndrea CalixtoXiaoyin

ChenYushu

ChenHongping

DuAnne DugganLesley EmtageGlen ErnstromGhia

EuskirchenJaime García-

AñoverosGuoqiang

GuMingxia

HuangSiavash

KarimzadeganCharles Keller

John KratzJoy LiangShujun

LuoCathy Savage Jim TaubJi

Wu Yi-Chun Wu Ding XueShifang

Zhang Yun

Zhang

Undergraduates

Macy Au Paul AuwaeterPeggy BrickmanKate BraumanJohn ByunAlex ChangVictor ChangIris ChinBradley CollinsMichael Dreyfuss

AngelaGeorgopoulos

Rachel GoldsteinJudith GreenPeter HomerRafaz

HoqueJames HudspethShari JawetzPaul JosephsonEric KanterLexy

KovachJoe LauSam LeeLorraine

LothringerDavid MeshoulamJeremy MindichMariya

RosenblitJulie RosenthalAaron SchefflerShai

ShahamJay SrinivasanLeslie Vosshall

Molly Weiner Chris William

Technicians

Margaret BarnesElke

BergholzLei Chen Ellen DeanNora HomMaud KinnellConnie MauokaEvelyn ReillyYuan TuYingzi

XueAndrea Yao

FacultyVisitors

Christian NeriJonathan

Rothblatt