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 migration hox genes 1, precursor cells 3, morphogenesis c ell division, fusion, migration, etc. Cell fate EEVVVE Lineage 2, vulval induction Cell signaling gonad P1P12 dorsal

AC 3° 2°1°2°3° WT X 3° - AC Indicating: A: AC is required for vulval induction B: AC may send a signal to induce vulval cells C: Both. Judith Kimble: gonad

AC 3° 2°1°2°3° WT X VPC ablation XXXXX 1°/2° Indicating: Paul Sternberg:

anchor cell inductive signal EEVVVE Wild type 100% induction EEE EEE Vulvaless 0% Multivulva200% VV V VVV signal pathwayfunction

- use genetics to get the major players in the pathway signalpathwayfunction

Earlier direct screens for vulval induction mutations wild type Mutagen Vulvaless Multivulva Horvitz and Sulston 1980, Genetics Ferguson and Horvitz 1985, Genetics Ferguson et al Nature Aroian et al Nature Hill and Sternberg Nature lin-3 EGF let-23 EGFR lin-1 Vulval induction

Identification of Ras Mammal: identified through oncogenic mutations in 1980 Ras homolog in yeast was identified in Ras function in development was first identified in 1990 (C. elegans) RAS GDP RAS GTP 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 Nature lin-15 Vulval induction Vote: X A. B. lin-15 Vulval induction X

Relationship between RAS and EGFR EGFR(lf)Vulvaless lin-15 Vulval induction EGFR RAS MultivulvaRAS (gf) EGFR(lf) + RAS (gf) Multivulva Vulval induction Vote: RAS A. B. Vulval induction EGFR RAS EGFR

Pathway in early 1992 RAS EGFR

RAS GDP RAS GTP Pi GTPGDP Exchange reaction Hydrolysis reaction target protein Active stateInactive Strategy 1: moving up from Ras signal

First strike: discovery of GTPase activating protein (GAP) 1. Questions addressed GTPase of Ras In vitroweakt 1/2 = 30 min In vivostrongt 1/2 < 1 min A: Something in cells can stimulate the GTPase activity B: 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 oocyte D. Yeast. RAS:GTP RAS: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 GDP Oncogenic 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:GTP RAS:GDP GTPase activity Pi RAS:GTP RAS:GDP GTPase activity Pi WT Gly 12 Oncogenes Val 12 Asp 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

RAS GDP RAS GTP Pi GTPGDP Exchange reaction GAP target protein Active stateInactive How is Ras regulated by the signal? EGFR

Cell It is all GAP Kaplan DR, Morrison DK, Wong G, McCormick F, Williams LT. Kaplan 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 : ReceptorGAP Ras Target

Doug Lowy: not so fast, GNEF is more important RAS GDP RAS GTP Pi GTPGDP Exchange reaction GAP target protein Active stateInactive EGFR

Ras (His116) mutant cause GDP to GTP exchange 10 x faster but 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 fast Adding EGF would have a small effect. (A. Yes, B. No) Results: model 1 should be right. RAS GDP RAS GTP Pi GTPGDP Exchange GAP Active stateInactive Zhang et al Science

The End