1. ~ 1000 cells, small, easy to use for genetics

2. Entire lineage and nerve system mapped.

Caenhorhabditis elegans

3. 3 day life cycle, easy to use for genetics.

4. hermaphrodite

Lineage by the John Sulston

The story of the lineage work

Upper Panel: Nomarski photomicrograph of one gonad arm of an adult C. elegans hermaphrodite. The distal portion of the gonad arm is up; the proximal portion is down.

Lower Panel: Nomarski photomicrograph of an adult C. elegans male gonad. The distal portion of the testis is above; the proximal region is below.

Using vulval development as a model system

Vulva: laying eggs mating

Reproductive system in C. elegans

Three steps of vulval development

cell migrationhox genes

1, precursor cells

3, morphogenesis cell division, fusion, migration, etc.

Cell fate E E V V V E

Lineage

2, vulval induction Cell signaling

gonadP1 P12dorsal

AC

3° 3° 2° 1° 2° 3°WT

X

3° 3° 3° 3° 3° 3°- AC

Indicating:

A: AC is required for vulval inductionB: AC may send a signal to induce vulval cellsC: Both.

Judith Kimble:

gonad

AC

3° 3° 2° 1° 2° 3°WT

XVPC ablation X X

X X X1°/2° 1°/2° 1°/2°

Indicating:

Paul Sternberg:

anchor cellinductive signal

E E V V V E

Wild type 100% induction

E E EEEE

Vulvaless 0%

Multivulva 200%

V V V V V V

signal pathway function

- use genetics to get the major players in the pathway

signal pathway function

Earlier direct screens for vulval induction mutations

wild type

Mutagen

Vulvaless

Multivulva

Horvitz and Sulston 1980, GeneticsFerguson and Horvitz 1985, GeneticsFerguson et al. 1987. NatureAroian et al. 1991. NatureHill and Sternberg 1992. Nature

lin-3EGF

let-23EGFR

lin-1

Vulval induction

Identification of Ras

Mammal: identified through oncogenic mutations in 1980

Ras homolog in yeast was identified in 1985.

Ras function in development was first identified in 1990 (C. elegans)

RASGDP

RASGTP

Pi

GTPGDP

Exchange reaction

Hydrolysis reaction

Active stateInactive

target protein

lin-15(-), Muv lin-15(-);; X(-) Vulvaless

EMS

Supressors of lin-15 mutation

X = EGFR, RAS and others. Han and Sternberg 1990, Cell; Beitel et al. 1990, Nature; Aroian et al. 1990 Nature

lin-15 Vulval induction

Vote:

XA.

B. lin-15 Vulval inductionX

Relationship between RAS and EGFR

EGFR(lf) Vulvaless

lin-15 Vulval inductionEGFRRAS

MultivulvaRAS (gf)

EGFR(lf) + RAS (gf) Multivulva

Vulval induction

Vote:

RASA.

B. Vulval induction

EGFR

RASEGFR

Pathway in early 1992

RASEGFR

RASGDP

RASGTP

Pi

GTPGDP

Exchange reaction

Hydrolysis reaction

target protein

Active stateInactive

Strategy 1: moving up from Ras

signal

signal

QuickTime™ and aGIF decompressor

are needed to see this picture.

QuickTime™ and aGIF decompressor

are needed to see this picture.

First strike: discovery of GTPase activating protein (GAP)

1. Questions addressed

GTPase of Ras

In vitro weak t 1/2 = 30 minIn vivo strong t 1/2 < 1 min

A: Something in cells can stimulate the GTPase activityB: Something in intro inhibits the GTPase activity

The student and PI were mammalian biochemists. What system will they likely chose to identify the “something”?

A: Mammalian cells. B. Drosophila. C. Xenopus oocyteD. Yeast.

RAS:GTPRAS:GDP

Pi

Approach: Using Xenopus oocytes- ease to manipulate due to large size- good assay for biological activity, oocyte maturation.

Does GTP or GDP bind to injected Ras?

WT Ras mostly bind to GDPOncogenic Ras mostly to GTP

In vivo In vitro

GTPase activity: speed in vivo

T 1/2 = 2-3 min for wild type. >1000 min for Asp12

WT vs. Oncogenes

RAS:GTPRAS:GDP

GTPase activityPi

RAS:GTPRAS:GDP

GTPase activityPi

WTGly 12

OncogenesVal 12Asp 12

X

What causes the difference between in vitro and in vivo?

Add cytoplasmic factor to in vitro > 200 fold difference

Trahey and McCormick Science Oct 1987

QuickTime™ and aGIF decompressor

are needed to see this picture.

RASGDP

RASGTP

Pi

GTPGDP

Exchange reaction

GAP

target protein

Active stateInactive

How is Ras regulated by the signal?

EGFR

EGFR

Cell 1990. It is all GAPKaplan DR, Morrison DK, Wong G, McCormick F, Williams LT. PDGF beta-receptor stimulates tyrosine phosphorylation of GAP and

association of GAP with a signaling complex. Cell. 1990 61:125-33.

Receptor GAP Ras Target

Doug Lowy: not so fast, GNEF is more important

RASGDP

RASGTP

Pi

GTPGDP

Exchange reaction

GAP

target protein

Active stateInactive

EGFR

EGFR

Ras (His116) mutant cause GDP to GTP exchange 10 x fasterbut its sensitivity to GAP is the same.

Model 1. EGFR GNEF RAS

Model 2. EGFR GAP RAS

If model 2 is right, GAP activity determines the GTPRas/GDPRas ratio add EGF, activity should dramatically increase. (A. Yes, B. No)

If model 1 is right, His 116 already already cause the exchange 10 X fastAdding EGF would have a small effect. (A. Yes, B. No)

Results: model 1 should be right.

RASGDP

RASGTP

Pi

GTPGDP

Exchange

GAPActive stateInactive

Zhang et al. 1991. Science

The End