Matias Simons, MD - Mount Sinai School of Medicine, New York

Wnt signaling and Polycystic kidney disease

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)

PKD is a ciliopathyPKD is a ciliopathy

All PKD gene products localize to the ciliumAll PKD gene products localize to the cilium

Kim and Walz, Nat Med 2007Piontek et al, Nat Med 2007

PKD is a developmental diseasePKD is a developmental disease

Perantoni, 2003

Wnt4

Wnt: key regulators of developmentWnt: key regulators of development

Overactivated canonical Wnt signaling

PKD

Saadi-Kheddouci et al, Oncogene 2001

Wnt signaling and PKDWnt signaling and PKD

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

canonical Wnt noncanonical Wnt- mesenchyme induction- cell proliferation - branching morphogenesis

switch

- tubular geometry?

- planar cell polarity?

Tubular flow

Simons and Walz, 2006

EPITHELIAL CELL WING EPITHELIAL CELL

Apical/Basal Polarity Planar Cell Polarity (PCP)

Epithelia exhibit two types pf polarityEpithelia exhibit two types pf polarity

PCP is evident in external epithelial tissuesPCP is evident in external epithelial tissues

Wild-type stbm/vangnull

vangl2Wild-type

M. Montcouquiol

PCP is conserved across species: cochleaPCP is conserved across species: cochlea

Canonical and noncanonical Wnt pathwaysCanonical and noncanonical Wnt pathways

Unknown activation step!!

Canonical and noncanonical Wnt pathwaysCanonical and noncanonical Wnt pathways

S2 cells

Dsh-GFP + FzDsh-GFP

Fz

CRD

DshDIX PDZ DEPb

S2 cells

Dsh-GFP + FzDsh-GFP

? ? ??

Fz

CRD

DshDIX PDZ DEPb

S2 cells

Dsh-GFP + FzDsh-GFP

? ? ??

Fz

CRD

DshDIX PDZ DEPb

S2 cells

Dsh-GFP + FzDsh-GFP

genome-wide RNAi screen in S2 cells

? ? ??

-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

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

H+

Na+

DmNhe2

DmNhe2 has highest sequence homology with mammalian Nhe3 -a plasma membrane exchanger

Main function: pH regulation

Sodium-proton exchangersSodium-proton exchangers

D.discoideum

alkaline pH microenvironments

asymmetric localization

oligodendrocytes

Nhe1

Nhe1

Plasma membrane exchangersPlasma membrane exchangers

PCP in the Drosophila eyePCP in the Drosophila eye

Nhe2 is involved in PCP signalingNhe2 is involved in PCP signaling

Nhe2 interacts genetically with FzNhe2 interacts genetically with Fz

Nhe2 interacts physically with FzNhe2 interacts physically with Fz

Reduction of intracellular pH affects Dsh recruitment

Reduction of intracellular pH affects Dsh recruitment

pH 7.1 pH 7.28pH 7.45

Dsh is surrounded by a higher pH at the membrane than in the cytosol

pH 7.45

pH 7.45

pH 7.71

Fz

CRD

DshDIX PDZ DEPb

Dsh DEP domainDsh DEP domain

Kd~10µM -> weak binding (Mol Cell, Wong et al, 2003)

Wong et al, 2000

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

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)

The polybasic stretch specifically interacts with PA

The polybasic stretch specifically interacts with PA

K/E: E E E E E

The polybasic stretch is important for Dsh recruitment by Fz in cells

The polybasic stretch is important for Dsh recruitment by Fz in cells

Same require-ments?

Dsh>Dsh KR/E-GFP X w, dshV26/FM7

Viability? PCP defects?

Dsh null fly rescue assayDsh null fly rescue assay

♂♂ ♀♀

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

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

Dsh recruitment modelDsh recruitment model

canonical Wnt noncanonical Wnt- mesenchyme induction- cell proliferation - branching morphogenesis

switch

- tubular geometry?

- planar polarity?

Tubular flow

canonical Wnt noncanonical Wnt- mesenchyme induction- cell proliferation - branching morphogenesis

switch

- tubular geometry?

- planar polarity?

Tubular flow

pH-dependent Dsh membrane recruitment

Marek Mlodzik+ lab membersMichael Boutros

(Heidelberg)Julian Dow

(Glasgow) Jie Zheng

(Memphis)Jie Chen

(Urbana)

Thanks toThanks to

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