Master class Biomolecular SciencesPart 1: Molecular Cell Biology.
October 9, 2008 Madelon Maurice
Department of Cell Biology, UMC, Utrecht ([email protected])
“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