Transcript Document

Caenhorhabditis elegans
1. ~ 1000 cells, small, easy to use for genetics
2. Entire lineage and nerve system mapped.
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
1, precursor cells
dorsal
gonad
P1
cell migration
hox genes
P12
2, vulval induction
Cell signaling
Cell fate
Lineage
3, morphogenesis
cell division, fusion,
migration, etc.
E
E
V
V
V
E
Judith Kimble:
AC
WT
3°
3°
2°
1°
gonad
2°
3°
3°
3°
X
- AC
3°
3°
3°
3°
Indicating:
A: AC is required for vulval induction
B: AC may send a signal to induce vulval cells
C: Both.
Paul Sternberg:
AC
WT
3°
3°
VPC ablation
1°/2°
Indicating:
1°/2°
2°
1°
2°
X
X
X
X
X
X
3°
1°/2°
anchor cell
inductive signal
E
E
V
V
Wild type
E
E
E
signal
V
E
100% induction
E
E
E
Vulvaless 0%
V
V
V
Multivulva
V
200%
V
V
pathway
function
signal
pathway
function
- use genetics to get the major players
in the pathway
Earlier direct screens for vulval induction mutations
Mutagen
Vulvaless
wild type
Multivulva
lin-1
lin-3
let-23
EGF
EGFR
Horvitz and Sulston 1980, Genetics
Ferguson and Horvitz 1985, Genetics
Ferguson et al. 1987. Nature
Aroian et al. 1991. Nature
Hill and Sternberg 1992. Nature
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)
GDP
Exchange
reaction
GTP
RAS
GDP
RAS
GTP
Inactive
target protein
Active state
Pi
Hydrolysis
reaction
Supressors of lin-15 mutation
EMS
lin-15(-);
; X(-) Vulvaless
lin-15(-), Muv
X = EGFR, RAS and others.
Han and Sternberg 1990, Cell; Beitel et al. 1990, Nature; Aroian et al. 1990 Nature
Vote:
A.
B.
lin-15
X
X
Vulval induction
lin-15
Vulval induction
Relationship between RAS and EGFR
lin-15
EGFR
RAS
Vulval induction
EGFR(lf)
Vulvaless
RAS (gf)
Multivulva
EGFR(lf) + RAS (gf)
Multivulva
Vote:
A.
RAS
B.
EGFR
EGFR
Vulval induction
RAS
Vulval induction
Pathway in early 1992
EGFR
RAS
Strategy 1: moving up from Ras
signal
Exchange
reaction
GDP
GTP
RAS
GDP
RAS
GTP
Inactive
Active state
Pi
signal
Hydrolysis
reaction
target protein
QuickTime™ and a
GIF decompressor
are needed to see this picture.
QuickTime™ and a
GIF decompressor
are needed to see this picture.
First strike: discovery of GTPase activating protein (GAP)
1. Questions addressed
RAS:GDP
GTPase of Ras
In vitro
In vivo
RAS:GTP
Pi
weak
strong
t 1/2 = 30 min
t 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.
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?
In vivo
WT Ras mostly bind to GDP
Oncogenic Ras mostly to GTP
In vitro
GTPase activity: speed in vivo
T 1/2 = 2-3 min for wild type.
>1000 min for Asp12
WT vs. Oncogenes
WT
Gly 12
RAS:GDP
Pi
Oncogenes
Val 12
Asp 12
RAS:GTP
GTPase activity
RAS:GDP
Pi
RAS:GTP
X
GTPase activity
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 a
GIF decompressor
are needed to see this picture.
How is Ras regulated by the signal?
EGFR
Exchange
reaction
GDP
GTP
RAS
GDP
RAS
GTP
Inactive
Active state
Pi
EGFR
GAP
target protein
Cell 1990. It is all GAP
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. 1990 61:125-33.
Receptor
GAP
Ras
Target
Doug Lowy: not so fast, GNEF is more important
EGFR
Exchange
reaction
GDP
GTP
RAS
GDP
RAS
GTP
Inactive
Active state
Pi
EGFR
GAP
target protein
Exchange
GDP
GTP
RAS
GDP
RAS
GTP
Inactive
GAP
Model 1. EGFR
GNEF
Model 2. EGFR
GAP
RAS
RAS
Active state
Pi
Ras (His116) mutant cause GDP to GTP exchange 10 x faster
but its sensitivity to GAP is the same.
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.
Zhang et al. 1991. Science
The End