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First published online 6 May 2008
doi: 10.1242/jcs.025536

Journal of Cell Science 121, 1793-1802 (2008)
Published by The Company of Biologists 2008
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Plasma membrane recruitment of dephosphorylated β-catenin upon activation of the Wnt pathway

Jolita Hendriksen1,*, Marnix Jansen1,2,*,{ddagger}, Carolyn M. Brown3, Hella van der Velde1, Marco van Ham4, Niels Galjart4, G. Johan Offerhaus2, Francois Fagotto3 and Maarten Fornerod1,§

1 Department of Tumor Biology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands
2 Department of Pathology, University Medical Center Utrecht, 3584 ZX Utrecht, The Netherlands
3 Department of Biology, McGill University,1205 Dr Penfield Avenue, Montreal, QC H3A 1B1, Canada
4 Department of Cell Biology, Erasmus MC, Dr Molewaterplein 50, 3015 GE Rotterdam, The Netherlands


Figure 1
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  		Fig. 1. Cadherin-independent plasma membrane localization of dephospho-β-catenin upon Wnt3A stimulation. (A) E-cadherin-negative cells respond normally to Wnt3a. Luciferase reporter assay in Kep1 (E-cadherin–/–) and Kp6 (E-cadherin+/+) cells using the TCF reporter TOP-TK and the control FOP-TK, normalized for transfection efficiency using pRL-CMV-Renilla. 24 hours after transfection, cells were stimulated overnight with Wnt3a-conditioned or control medium and luciferase activity was measured. (B) (Dephospho) β-catenin levels in Kep1 or Kp6 cells. Cells were induced with Wnt3A protein or control and analyzed 0.5 or 2.5 hours after induction by western blotting using an antibody recognizing all forms of β-catenin (TCAT) or an antibody specific for the N-terminal dephospho form (ABC). Asterisk indicates a crossreacting protein. (C) Subcellular localization of dephospho-β-catenin in E-cadherin positive or negative cells upon Wnt stimulation. Kep1 (E-cadherin–/–) or Kp6 (E-cadherin+/+) cells were induced with Wnt3A protein or control and analyzed 2.5 hours after induction by immunolocalization with an antibody specific for the N-terminal dephospho form (ABC). DAPI was used as a nuclear marker. Note that dephospho-β-catenin levels in E-cadherin–/– cells are much lower than in E-cadherin+/+ cells, requiring unequal confocal settings to be used.

 

Figure 2
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  		Fig. 2. Membrane recruitment of dephospho-β-catenin is an early event in the Wnt response. Kep1 cells were induced with Wnt3A for 30-240 minutes and nuclear and (dephospho-)β-catenin plasma membrane accumulation was recorded in 100-130 cells (see Materials and Methods for details on image acquisition and analysis). (A) Representative fields of cells. (B) Box plots representing quantification of plasma membrane and nuclear accumulation. Medians are indicated in red. P values indicated are the results of Mann-Whitney tests.

 

Figure 3
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  		Fig. 3. Dephospho-β-catenin colocalizes with p-LRP6, axin and APC at the plasma membrane in a Wnt-dependent (Kep1) or {Delta}N-LRP6-dependent (SK-BR-3) manner. Kep1 cells were induced with purified Wnt3A protein or vehicle for 2.5 hours (Kep1), or transfected with {Delta}N-LRP6-encoding plasmid (SK-BR-3) and analyzed for subcellular localization of dephospho-β-catenin and phospho-LRP6 (A,D), dephospho-β-catenin and axin (B), total β-catenin and APC (C), and dephospho-β-catenin and APC (E). Arrows denote plasma membrane localizations. Merged images are also shown, which include DAPI as a nuclear marker (A-C) or H2B-mRFP as a transfection marker (D,E).

 

Figure 4
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  		Fig. 4. LRP6-initiated dephospho-β-catenin is transcriptionally more active than downstream-initiated dephospho-β-catenin. Cadherin-deficient SK-BR-3 breast carcinoma cells were transiently transfected with 25 or 50 ng plasmid encoding wild-type β-catenin or 160 ng of a plasmid encoding {Delta}N-LRP6 and analyzed by immunolocalization (A,B), TCF transcriptional activity (C) and western blotting (D,E). (A,B) Immunolocalization of dephospho-β-catenin in cells exogenously expressing β-catenin or {Delta}N-LRP6, identified by coexpression of mRFP-tagged histone H2B. (C) TCF-dependent transcriptional activity in SK-BR3 cells transfected with indicated plasmids 24 hours after transfection. TOP, TCF-reporter luciferase activity; FOP, mutated TCF-reporter activity. Values were normalized to a transfection control (constitutive Renilla luciferase reporter). (D,E) Western blot analysis of cells shown in A-C, detecting total (D) or dephospho-β-catenin (E) levels. 4x, fourfold amount loaded; 0.5x, one half amount loaded. Asterisk indicates a cross-reacting protein.

 

Figure 5
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  		Fig. 5. Supraphysiological levels of exogenous β-catenin are required to mimic Wnt activity in Xenopus embryos. β-catenin (1.0 ng) or Wnt8 (50 pg) mRNA was injected into the ventral side of four-cell-stage embryos and total β-catenin levels achieved at blastula stage were estimated by immunofluorescence on cryosections. (A) Diagram of cross-section of a blastula embryo indicating the areas used to compare β-catenin staining. (B,C) Ventral and dorsal regions of uninjected embryos. Arrows indicate nuclear β-catenin and arrowheads, the plasma membranes. (D) Ventral region from Wnt8-injected embryo. Note the increased nuclear signal. (E) Ventral region from β-catenin-injected embryo. Note the very strong signal throughout the cells. (F) The same field recorded with a five times shorter exposure time. (G) Comparison of dorsalizing activity of Wnt and β-catenin in early Xenopus embryos. The degree of dorsalization obtained was scored at the tailbud stage. The phenotypes were classified into five categories of increasing dorsalization: normal; partial duplicated axis; complete duplicated axis; complete duplicated axis with shortened axis indicating partial global dorsalization; and completely dorsalized, i.e. global dorsalization with reduced or no axis and radial head structures. P value according to {chi}2 test.

 

Figure 6
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  		Fig. 6. Membrane localization of dephospho-β-catenin in the early Xenopus embryo. Cryosections of stage 9 embryos were stained for dephospho-β-catenin (ABC) and total β-catenin (H102) and nuclei were counterstained with DAPI. (A) Selected fields of the ventral presumptive ectoderm and mesoderm (uninjected ventral), dorsal mesoderm (uninjected dorsal), and ventral ectoderm of an embryo injected with 50 pg Wnt8 mRNA (Wnt-injected). In uninjected embryos, ABC stains the outlines of mesodermal cells (me) (arrowheads) but not ectodermal cells (ec) (arrows). Membrane ABC is detected in ectodermal cells of Wnt-injected embryos (arrowheads). Small bright cytoplasmic spots in all three color channels correspond to autofluorescent pigment granules. (B) Higher magnification view of ABC staining of ectodermal (ec) and mesodermal (me) cells of uninjected cells. These two images were obtained by collection of z-stacks followed by 2D nearest neighbors deconvolution and merging 10 images from each stack.

 

Figure 7
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  		Fig. 7. Dephospho-β-catenin is enriched on the plasma membrane in human intestinal crypts. (A) Low-power photomicrograph of a normal human small intestinal crypt-villus axis. Dephospho-β-catenin is enriched on the crypt epithelial plasma membrane when compared with the plasma membrane labeling on differentiated villus cells. (B) High-power photomicrograph of crypt compartment shown in A. Crypt epithelial cells including the presumptive intestinal epithelial stem cells or crypt base columnar (CBC) cells (Barker et al., 2007Go) show robust plasma membrane labelling (arrows).

 

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