Master class Biomolecular SciencesPart 1: Molecular Cell Biology.

October 9, 2008 Madelon Maurice

Department of Cell Biology, UMC, Utrecht (M.M.Maurice@umcutrecht.nl)

“Cell Biology of Wnt signaling”

Article:

Franch-Marro X, Wendler F, Guidato S, Griffith J, Baena-Lopez A, Itasaki N, Maurice MM, Vincent JP (2008) Wingless secretion requires endosome-to-Golgi retrieval of Wntless-Evi-Sprinter by the retromer complex. Nat Cell Biol 10(2):170-77

Evolutionary conservation of the Wnt gene family.

Evolutionary conservation of the Wnt gene family.

Wnt homepage, R.Nusse

(Sea urchins)

(flat worms)

(jelly fish)

The Wnt signalling cascade

Wnt

TCF TCF

P

Destructioncomplex

-cat

-catenin

TranscriptionNo transcription

Destructioncomplex

Ub

Proteasomal degradation

Fz Lrp5/6

Wnt signalling:- Development (body axis, tissue patterning) and adult tissue homeostasis- Cell growth, lineage decisions, stem cell maintenance- Frequent target for mutations in cancer

OFF ON

?

A. Gregorieff, Gen Dev 2005

Adult intestinal homeostasis

KI67

TCF TCF

P

Destructioncomplex

-cat

-cat

TranscriptionNo transcription

Destructioncomplex

The Wnt signalling cascade

Ub

Proteasomal degradation

OFF CANCER

Strutt, D. Development 2003;130:4501-4513

-catenin dependent and independent Wnt signalling pathways

Small GTPases,JNK signaling

Wnt/-catenin

Strutt, D. Development 2003;130:4501-4513

Planar polarity and Fz phenotypes in the Drosophila wing

Pupal stage

Wnt family: - 19 members in man and mice- ~ 40kD- Secreted Cys-rich glycoproteins- Lipid-modified

Wnts - Introduction

Wnt functions: - Embryogenesis and homeostasis of self- renewing tissues

- Mitogenic stimulation - Cell fate specification / lineage decisions - Stem cell maintenance

Palmitoleic acid

SerWg

Palmitate

Wnts act as morphogens

Wnt secretion

Spread: 20-25 cell diameters

Morphogen: - Secreted, diffusable signalling molecules - Generate discrete genetic programs in a concentration-dependent manner (positional information)

Short-range vs. long-range signaling: The ‘hydrophobicity problem’

Wnt (Wg) gradient is stable and has defined steepness.

How is this accomplished?

Three factors proposed to contribute:1. Rate of production/secretion2. Rate of spreading3. Rate of degradation

Models of gradient formation1. Cellular inheritance2. Diffusion and lysosomal degradation3. Planar transcytosis

Extracellular components of Wnt (Wg) gradient formation

wt

mut

mut

mut

A. Cellular inheritance(gradient established = 1 hour, avg doubling time cells = 11 hours)

Models for Wnt gradient formation

B. Diffusion and lysosomal degradation

C. PLanar transcytosis(gradient forms extracellularly,Endocytosis does not affectWg transport)

apical

basal(Secretion apical, gradient forms basolaterally)

*

*

*

*

*

Models proposed:1. Multimeric complexes - Wnt ‘micelles’ 2. Vesicles (exosomes) - MVB derived3. Membrane protrusions - imaginal disc cells construct

apical protrusions: cytonemes4. Lipoprotein particles - phospholipid monolayers

surrounding a core of esterified cholesterol and triglyceride, scaffolded by apolipoproteins

- 10% Wg bound, required for long- range Wg signaling

What is the nature of secreted Wnt (Wg) particles?

Two-gradient model: explaining short- and long-range Wnt signaling

- Lipoprotein particles

(membrane association)

(Transcytosis needed)

Where in the cell is Wnt lipid-modified?

How is Wnt inserted into lipoprotein particles?

In what subcellular compartment does this take place?

Does Wnt trafficking and secretion require specialized machinery?

How do short-range signals differ from long-range signals?

Questions

1. Porcupine (EMBO 1993) - Acyl transferase, palmitoylation of Wnt- ER resident protein, 8-span TM, highly

conserved- porc mutant (D.m.):

retention of Wg in producing cells- Required for proper N-

glycosylation and secretion of Wnt

2. Retromer complex (Science 2006) - Recycling of proteins/receptors from

endosome to TGN- Mutant (C.e/D.m.): No secretion

3. Wntless/Evi (Cell 2006) - 7-span transmembrane molecule- Localizes to Golgi and PM- Mutant (C.e./D.m.): No secretion, accum.

Wnt in producing cells

Intracellular molecular machinery involved in Wg secretion

anterior posterior

Retromermutant

Wnt gradient formation requires retromer function in Wnt-producing cells

Coudreuse et al. (2006) Science 312:p921

-cat mutant

Nucleus / ER

Golgi

Trans-Golgi Network

Early Endosome

Late Endosome

Lysosome

TEN/TSE

Retromer

Plasma membrane

Cellular function of the retromer complex

Sec

reto

ry p

ath

wayD

egrad

ative path

way

Endocytosis

The retromer complex: organization and function

Seaman, Trends in Cell biology, 2005

SNX1 and 2: PH- and BAR-domain proteinsVps26/29/35: cargo selectionVps35: binds cargo

? ?

What cargo is transported by the retromer complex?

N-ter SNX

No structuralinformation

working model for the assembly and function of mammalian retromer

Example retromer-cargo: Mannose-6-phosphate receptor(CD-MPR)

Retromer

= Lysosomalhydrolases

Binds to clathrin, concentrates MPRs

?? Budding?

Tethering?Fusion?

What is the relationship between Evi/Wls and retromer?

Common stages to look for effects of Wg signaling:Patterning epidermis

Drosophila as a model system for studying the Wnt (Wg) pathway

Wing disc

Wing margin

(400/female)

- Larvae molt twice, ecdysis- Eat microorg on decaying fruit and fruit-derived sugar

Larvae encapsulatein puparium; metamorphosis

Expression of wingless (Wg) and vestigial in the Drosophila wing disc

Wg = greenVestigial = red

D/V boundary

Wing disc expressing C93A HA-Wg (Dpp Gal4 –driven)

Fixation 2% PFA, 0.2%GA

300x

pos

neg

Disc cells strongly expressing C93A HA-Wg are grouped together

HA-Wg15 nm

Peripodial membrane

Disc cells expressing C93A HA-Wg are grouped together

C93AHA-Wg15

Peripodial lumen

Peripodial membrane

Common stages to look for effects of Wg signaling:Patterning epidermis

Drosophila as a model system for studying the Wnt (Wg) pathway

Wing disc

Wing margin

Epidermal patterning of Wg in fly embryo

Common stages to look for effects of Wg signaling:Patterning epidermis

Drosophila as a model system for studying the Wnt (Wg) pathway

Wing disc

Wing margin

notum

wing hinge

wing blade

wing margin

The wing disc and its adult derivatives

D V