drosophila
DESCRIPTION
variation. Drosophila. Egg chamber. Eggshell (chorion). ?. ?. Y*. The experimental system. Cellular responses. EGFR/ErbB Signaling Network. Invertebrates. Vertebrates. ErbB3. EGFR. ErbB2. EGFR. signal diversification • inhibitors ?. signal diversification: • heterodimerization. - PowerPoint PPT PresentationTRANSCRIPT
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DrosophilaEgg chamber Eggshell (chorion)
variation
??
The experimental system
Y*Cellular responses
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EGFR/ErbB Signaling Network
Vertebrates
EGFR ErbB2 ErbB3
Invertebrates
EGFR
signal diversification• inhibitors ?
signal diversification:• heterodimerization
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The EGFR network and dorsal patterningThe EGFR network and dorsal patterning
Profile of EGFR activity: WTInitiation
Gurken
InhibitionArgos
AmplificationSpitzVein
Site ofAppendageFormation
chorion
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How can we identify components of the EGFR network?
identify targets
Molecular
Create differences = mutant screens
Genetic Phylogenetic
Identify differences = other species
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kek1 expression
Kek1EGFR
ligand
Kek1 and feedback regulation of EGFR
?No overt phenotype ?
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Kek1EGFR
ligand
Kek1 overexpression inhibits receptor signaling
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Evidence that endogenous kek1 inhibits EGFR signaling
WT kek1-
aos
rho
BR-C
WT egfr- egfr-; kek1-
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InitiationGurken
AmplificationSpitzVein
InhibitionArgos
Site ofAppendageFormation
What role does Kek1 have in dorsal patterning?What role does Kek1 have in dorsal patterning?
Profile of EGFR activity: WT
Profile of EGFR activity: kek1-
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EGFRKek1EGFR
ligand
1. Mechanism2. Sequence requirements
Kek1 inhibits EGFR signaling
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EGFR
Ligand sink vs heterodimer
1. Mechanism
Kek1 inhibits EGFR signaling
Kek1
No signaling - ventralized
Kek1
Y*
Signaling - dorsalized
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Kek1 inhibits EGFR signaling
EGFRKek1
2. Sequence requirements ?
LRRs
Ig
N FLANK
C FLANK
Kek2Kek5
Kek6Kek4Kek3
Kek1
EC
IC
Domain swaps
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2. Sequence requirements ?
Kek1 inhibits EGFR signaling
EGFRKek1
Kek1 Kek2LLILIT
160
75
180
IP: anti-GFPIB: anti-EGFR
IP and IB: anti-GFP
IB: anti-EGFR
KDa
180
K1 LIT LI L K2K1 K2
The Kek1 LRRs are sufficient for binding EGFR…
L
LIT
…but insufficient for full inhibition in vivo!
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____________________CyO
[GMR-GAL4] [UAS-kek1gfp]
suppressor
____________________+
[GMR-GAL4*] [UAS-kek1*gfp]
EMS
w; iso2; iso3
P
F1
Identifying residues important for Kek1/EGFR interaction
EMS
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Knockouts of Kek1
Kek1
L136F
G160S
G160D
P329S
IP: anti-GFPIB: anti-EGFR
IP and IB: anti-GFP
IB: anti-EGFR
180 KDa
55
180
160
70
EGFR + + + + +WT
180 KDa
180 KDa
160 KDa
IP GFP
IB -EGFR
Cell lysate IB:-EGFR
IP and IB: GFP
wt SOK3 SOK4 wt SOK3 SOK4 wt SOK3 SOK4EGFR
Kek1-GFP EGFR-GFP KECG
Suppressors of Kek1= egfr alleles
Identifying residues important for Kek1/EGFR interaction
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EGFRKek1
Kek1 = a novel inhibitory component of the EGFR network
Trafficking &
Stability
Binding&
Inhibition
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Identification of EGFR components Identification of EGFR components Genetic approach
WT bwkdCBP
Directed mosaics
DrosophilaEgg chamberMutations in
vital gene =lethalityX
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dCBP mediates feedback inhibition of EGFR signaling dCBP mediates feedback inhibition of EGFR signaling
wild type EGFR dCBP pnt
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InitiationGurken
AmplificationSpitzVein
InhibitionArgos
Site ofAppendageFormation
chorion
wt dCBP -
dCBP mediates feedback inhibition of EGFR signaling dCBP mediates feedback inhibition of EGFR signaling
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Egfr mediated epithelial Egfr mediated epithelial patterningpatterning
EGFR
Egfr transcriptional targets
Bwk
amplification
Rhomboid Kek1
attenuation
dCBP/Pointed
Argos
inhibition
Gurken
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latifasciaformis
Identification of EGFR components Identification of EGFR components
L.Throckmorton, 1950
1144
33
44
2222
22
Phylogenetic approach
44
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map natural variation
? genes
Interspecificanalysis
dose-dependent screen
EGFR signaling and appendage patterning
Intraspecificanalysis
Characterize patterningat molecular level
What components of the pathway underlie variation in dorsal appendage patterning?
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A
B
C
D
E
F
G
H
I
JD. melanogaster D. virilis
Interspecific analysis: Kek1
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grkHK36 egfrCO
+ +
Appendage variation
WT/V0 V1a V1b V2 V3 V4
• Does quantitative variation in the EGFR network exist?• If so, what molecular components underlie this variation?
- template for interspecific variation?
Intraspecific analysis
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Intraspecific variation in EGFR signaling
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EGFR signaling and appendage patterning
grkHK36 egfrCO
+ +
Identifying novel dose-dependent
EGFR components
0
5
10
15
20
25
30
35
40
45
50
# of Df's
>75% 51-75% 25-50% <25%% WT chorions
Screen for modifiers of EGFR signaling
3
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Egfr mediated epithelial Egfr mediated epithelial patterningpatterning
EGFR
Egfr transcriptional targets
Bwk
amplification
Rhomboid Kek1
attenuation
dCBP/Pointed
Argos
inhibition
Gurken
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Diego AlvaradoAlex DoetschTim EvansChristina MacLarenAmy RiceAnne Scherer Fred Derheimer
Libbie EakinPaul MillerChristopher SkipwithBrandon Weasner
Andrea CowdenOmar HasnieMatt RubinKyle Payne