First published online 1 November 2005
doi: 10.1242/jcs.02647
Journal of Cell Science 118, 5279-5289 (2005)
Published by The Company of Biologists 2005
Dishevelled (Dvl-2) activates canonical Wnt signalling in the absence of cytoplasmic puncta
Matthew J. Smalley1,
Nathalie Signoret2,
David Robertson1,
Alan Tilley3,
Anthony Hann5,
Ken Ewan5,
Yanning Ding4,
Hugh Paterson4 and
Trevor C. Dale5,*
1 The Breakthrough Breast Cancer Research Centre, The Institute of Cancer Research, 237 Fulham Road, London, SW3 6JB, UK
2 MRC Cell Biology Unit-LMCB, University College London, Gower Street, London, WC1E 6BT, UK
3 Improvision, Viscount Centre II, University of Warwick Science Park, Milburn Hill Road, Coventry, CV4 7HS, UK
4 CRC Centre for Cell and Molecular Biology, The Institute of Cancer Research, 237 Fulham Road, London, SW3 6JB, UK
5 Cardiff School of Biosciences, Biomedical Sciences Building, Museum Avenue, Cardiff, CF10 3US, UK
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Fig. 1. Localisation of ectopically expressed Dvl-2. (A-D) Localisation of transiently expressed HA-Dvl-2-EGFP (green) in HEK293 cells (A-B, nuclei counterstained with ToPro III), MDCK cells (C, nuclei counterstained with ToPro III) and CHO cells (D, no nuclear counterstain). (E-F) Localisation of transiently expressed HA-Dvl-2-ER (green) in MDCK cells. Cells were fixed 1 hour after re-feeding with medium containing ethanol vehicle (E) or 1 µM ß-estradiol (F). Bar, 15 µm.
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Fig. 3. Activation of canonical Wnt signalling by ectopic expression of Dvl-2 in high- and low-level expression systems. (A) HEK293 cells were transiently transfected (`high-level ectopic expression system') with either TCF-dependent transcription responsive (`canonical' Wnt signalling) reporter plasmids, reporter plasmids plus HA-Dvl-2 or reporter plasmids plus HA-Dvl-2-ER. Cultures were re-fed with medium containing 1 µM estradiol or control medium 24 hours prior to analysis for luciferase activity and protein levels in the detergent soluble and insoluble fractions of the cell extracts. (B) Time course of activation of canonical Wnt signalling pathway in Dvl-2-ER single-copy cell line. The cells were re-fed with medium containing 1 µM estradiol or with control medium and then harvested after 10, 30 and 60 minutes (10m/30m/60m) and 24 hours (24h) of induction. Detergent-soluble and -insoluble fractions were analysed by SDS-PAGE and western blotting. Identical protein levels were run on the gels. Blots were probed for the HA-tagged exogenous Dishevelled, for endogenous and exogenous Dvl-2 with a rabbit polyclonal anti-Dvl-2 and for endogenous ß-catenin and GSK-3ß. Note that although GSK-3ß levels remain constant, there was an increase in soluble ß-catenin levels from 30 minutes after ß-estradiol treatment. Also, note the disappearance of the endogenous Dvl-2 protein (anti-Dvl-2 probe, 98kD band) from the soluble fraction following ß-estradiol treatment, paralleling that of the single-copy fusion protein (150 kDa band). Finally, note the similarity in expression levels of the endogenous and exogenous proteins (estimated at three- to fivefold higher for the fusion protein). (C) Analysis of ß-catenin and endogenous Dvl-2 expression in the parental cell line after 60 minutes (60m) and 24 hours (24h) of ß-estradiol treatment. Note that there was no change in expression levels.
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Fig. 4. Co-staining of HA-Dvl-2-EGFP with endocytic markers. (A-F) HEK293 cells immunostained with antibodies against endogenous EEA-1 (A,B), transferrin receptor (C,D) and CD63 (E,F). Antibody localisation is shown in red. There was no non-specific background staining when control isotype-specific antibodies was used as the primary antibody (data not shown). Nuclei were counterstained with ToPro III (blue). A,C,E, non-transfected cells. B,D,F, cells transfected with HA-Dvl-2-GFP. (G-H) Localisation of Alexa-594-coupled transferrin in HA-Dvl-2-GFP-transfected CHO cells, identifying peripheral transferrin-containing vesicles and early endosomes (G) or perinuclear recycling endosomes (H). (I) Localisation of late endosomes in HA-Dvl-2-GFP transfected CHO cells using an antibody to LgpB. Note that HA-Dvl-2-GFP failed to colocalise with any of these markers. (J) Analysis of HA-Dvl-2-GFP puncta using electron microscopy of HA-Dvl-2-GFP transfected HEK293 cells. The nuclear membrane (arrowheads) is clearly seen around the nucleus (n). In contrast, there is no evidence of a membrane around the electron-dense body (p), in which the HA-Dvl-2-GFP is concentrated, as shown by the anti-HA immuno-gold labelling in the magnified inset box (arrows). Bars, 5 µm (A-F); 20 µm (G-I); 1 µm (J).
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Fig. 7. Time-lapse analysis of HA-Dvl-2-EGFP puncta. (A) Still frames from Movie 1B in supplementary material, illustrating lack of coordinated or directional movement in fluorescent bodies in a cell ectopically expressing HA-Dvl-2-EGFP at high levels. Frame numbers are indicated. Four bodies are false-coloured in the first frame (213) so that their fate may be followed. Note the yellow and green bodies fuse in frame 230 and the orange and red bodies fuse via a `dumb-bell' intermediate in frame 263. (B) Still frames from Movie 1C in supplementary material, illustrating lack of coordinated or directional movement in fluorescent bodies in a cell expressing lower levels of HA-Dvl-2-EGFP. Frame numbers are indicated. Two bodies are false coloured in the first frame (256) so that their fate may be followed. Note the red body appeared to split into two between frames 293 and 308, the yellow body disappeared by frame 314 and one of the two bodies resulting from the split of the red body disappeared by frame 328. Bars, 12 µm.
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© The Company of Biologists Ltd 2005
-tubulin and HA-Dvl-2-ER protein with 
-tubulin antibody confirmed that microtubule networks were disrupted only in nocodazole-treated cultures and were not affected by the fixation protocol (data not shown). Similar disruption of HA-Dvl-2-ER at the membrane was seen in cells treated with nocodazole for periods of between 2 hours and 15 minutes (data not shown). Bars, 10 µm.
DIX HA-Dvl-2-ER vehicle only. (B) 