Wnt signaling and Polycystic kidney disease - HÔPITAL …nephro-necker.org/pdf/2008/Simmons.pdf ·...
Transcript of Wnt signaling and Polycystic kidney disease - HÔPITAL …nephro-necker.org/pdf/2008/Simmons.pdf ·...
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Matias Simons, MD - Mount Sinai School of Medicine, New York
Wnt signaling and Polycystic kidney disease
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Most common monogenic disease
1/500 in the population
10% of end-stage renal disease
slowly progressive genetic disorder
different types: ADPKD, ARPKD, nephronophthisis
Polycystic kidney disease (PKD)Polycystic kidney disease (PKD)
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PKD is a ciliopathyPKD is a ciliopathy
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All PKD gene products localize to the ciliumAll PKD gene products localize to the cilium
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Kim and Walz, Nat Med 2007Piontek et al, Nat Med 2007
PKD is a developmental diseasePKD is a developmental disease
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Perantoni, 2003
Wnt4
Wnt: key regulators of developmentWnt: key regulators of development
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Overactivated canonical Wnt signaling
PKD
Saadi-Kheddouci et al, Oncogene 2001
Wnt signaling and PKDWnt signaling and PKD
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Inversin, BBS4, KIF3a - ciliary proteins that inhibit canonical wnt pathway
Simons et al, Nat Gen, 2005Gerdes et al, Nat Gen, 2007Corbit et al, Nat Cell Biol 2007
Cilia inhibit canonical Wnt signalingCilia inhibit canonical Wnt signaling
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canonical Wnt noncanonical Wnt- mesenchyme induction- cell proliferation - branching morphogenesis
switch
- tubular geometry?
- planar cell polarity?
Tubular flow
Simons and Walz, 2006
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EPITHELIAL CELL WING EPITHELIAL CELL
Apical/Basal Polarity Planar Cell Polarity (PCP)
Epithelia exhibit two types pf polarityEpithelia exhibit two types pf polarity
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PCP is evident in external epithelial tissuesPCP is evident in external epithelial tissues
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Wild-type stbm/vangnull
vangl2Wild-type
M. Montcouquiol
PCP is conserved across species: cochleaPCP is conserved across species: cochlea
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Canonical and noncanonical Wnt pathwaysCanonical and noncanonical Wnt pathways
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Unknown activation step!!
Canonical and noncanonical Wnt pathwaysCanonical and noncanonical Wnt pathways
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S2 cells
Dsh-GFP + FzDsh-GFP
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Fz
CRD
DshDIX PDZ DEPb
S2 cells
Dsh-GFP + FzDsh-GFP
? ? ??
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Fz
CRD
DshDIX PDZ DEPb
S2 cells
Dsh-GFP + FzDsh-GFP
? ? ??
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Fz
CRD
DshDIX PDZ DEPb
S2 cells
Dsh-GFP + FzDsh-GFP
genome-wide RNAi screen in S2 cells
? ? ??
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-Hel25E
-SF2
-Snap
−δCOP-Sec61�
-Fz
-Par-1
-CG11297
-Nhe2
-PebIII
-CG15237
-CG15243
-CG14756
-CG7519
RNAi screen candidatesRNAi screen candidates
RNA factors
trafficking factors
controls
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Control RNAi Nhe2 RNAi
-CG11297
-Nhe2-PebIII
-CG15237
-CG15243
-CG14756
-CG7519
-Hel25E
-SF2
-Snap
−δCOP-Sec61�
-Fz
-Par-1
RNAi screen candidatesRNAi screen candidates
RNA factors
trafficking factors
controls
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H+
Na+
DmNhe2
DmNhe2 has highest sequence homology with mammalian Nhe3 -a plasma membrane exchanger
Main function: pH regulation
Sodium-proton exchangersSodium-proton exchangers
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D.discoideum
alkaline pH microenvironments
asymmetric localization
oligodendrocytes
Nhe1
Nhe1
Plasma membrane exchangersPlasma membrane exchangers
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PCP in the Drosophila eyePCP in the Drosophila eye
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Nhe2 is involved in PCP signalingNhe2 is involved in PCP signaling
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Nhe2 interacts genetically with FzNhe2 interacts genetically with Fz
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Nhe2 interacts physically with FzNhe2 interacts physically with Fz
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Reduction of intracellular pH affects Dsh recruitment
Reduction of intracellular pH affects Dsh recruitment
pH 7.1 pH 7.28pH 7.45
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Dsh is surrounded by a higher pH at the membrane than in the cytosol
pH 7.45
pH 7.45
pH 7.71
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Fz
CRD
DshDIX PDZ DEPb
Dsh DEP domainDsh DEP domain
Kd~10µM -> weak binding (Mol Cell, Wong et al, 2003)
Wong et al, 2000
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The DEP domain binds to acidic phospholipids
The DEP domain binds to acidic phospholipids
SUV-liposome bound Free protein
Jie Zheng (St. Jude, Memphis)Jie Chen (UIUC,Urbana)
neg. charged
neutral
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The polybasic stretch interacts with PA in an electrostatic manner
The polybasic stretch interacts with PA in an electrostatic manner
K/E: E E E E E
SUV-liposome bound Free protein
Jie Zheng (St. Jude, Memphis)Jie Chen (UIUC,Urbana)
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The polybasic stretch specifically interacts with PA
The polybasic stretch specifically interacts with PA
K/E: E E E E E
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The polybasic stretch is important for Dsh recruitment by Fz in cells
The polybasic stretch is important for Dsh recruitment by Fz in cells
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Same require-ments?
Dsh>Dsh KR/E-GFP X w, dshV26/FM7
Viability? PCP defects?
Dsh null fly rescue assayDsh null fly rescue assay
♂♂ ♀♀
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dshV26; dsh>dsh-GFP KR/E
dshV26; dsh>dsh-GFP wt
The polybasic stretch is important for Fz/PCP signaling
The polybasic stretch is important for Fz/PCP signaling
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SummarySummary
• Nhe2 knockdown impairs Dsh recruitment in S2 cells• Nhe2 gain-of and loss-of-function causes PCP defects in
vivo• Nhe2 genetically and physically interacts with Fz• Dsh membrane recruitment is disrupted by low pH• Dsh is surrounded by a higher pH at the membrane • Dsh DEP domain specifically binds to phosphatidic acid in
the membrane via a polybasic stretch• The polybasic stretch is necessary for Dsh recruitment to
the plasma membrane by Fz• The polybasic stretch is important for Fz/PCP signaling in
vivo
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Dsh recruitment modelDsh recruitment model
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canonical Wnt noncanonical Wnt- mesenchyme induction- cell proliferation - branching morphogenesis
switch
- tubular geometry?
- planar polarity?
Tubular flow
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canonical Wnt noncanonical Wnt- mesenchyme induction- cell proliferation - branching morphogenesis
switch
- tubular geometry?
- planar polarity?
Tubular flow
pH-dependent Dsh membrane recruitment
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Marek Mlodzik+ lab membersMichael Boutros
(Heidelberg)Julian Dow
(Glasgow) Jie Zheng
(Memphis)Jie Chen
(Urbana)
Thanks toThanks to
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Thanks to
Marek MlodzikWilliam GaultUrsula WeberDavid del AlamoJun WuMei-Ling ChinEkatherina SheryshevaJaskirat SinghSophie OkelloGustavo GarciaJean-Remy CourbardPatricio OlguinAlex Wang
Former members:Andreas JennyThomas Klein
Collaborators:
Michael Boutros
Jie Zheng
Jie Chen
Julian Dow
Daniel Gotthardt