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First published online 1 November 2005
doi: 10.1242/jcs.02646

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The Wnt signalling effector Dishevelled forms dynamic protein assemblies rather than stable associations with cytoplasmic vesicles

MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, UK

View larger version (20K): [in a new window]   Fig. 1. Dvl2 puncta in transfected mammalian cells. COS-7 cells expressing HA-Dvl2 were fixed and stained with anti-HA antibody at the following time-points after transfection: (A) 18 hours; (B) 24 hours; (C) 36 hours; (D) 48 hours. Bar, 15 µm.

View larger version (57K): [in a new window]   Fig. 2. Correlation between Dvl2 puncta formation and signalling activity. COS-7 cells expressing (A) wild-type, (B) M68/69 or (C) DIX mutant HA-Dvl2, were fixed and stained as in Fig. 1. Notice that neither mutant is able to form puncta (see also Capelluto et al., 2002). (D) Wnt signalling activity of wild-type and M68/69 mutant HA-Dvl2 in transfected 293T cells, as measured by TOPFLASH transcription assays (see Materials and Methods). Numbers underneath bars represent µg of DNA used for transfection (1, 1 and 3 µg). Protein expression levels were confirmed by western blotting (not shown). Bar, 15 µm.

View larger version (114K): [in a new window]   Fig. 3. Dvl2 puncta do not overlap significantly with conA fluorescence. COS-7 cells expressing HA-Dvl2 were fixed and stained as in Fig. 1 after incubation with fluorescently-labelled conA for 45 minutes; two representative cells are shown. Arrows indicate rare yellow signals that reflect coincidental green (Dvl2) and red (conA) fluorescence. Bars, 15 µm.

View larger version (130K): [in a new window]   Fig. 4. Lack of colocalisation between Dvl2 puncta and endocytic markers. COS-7 cells expressing HA-Dvl2, were fixed and double-stained with antibodies against HA (green), and various endocytic markers (red) as indicated. (A-C) Endogenous endocytic and Golgi proteins; (D-F) exogenous endocytic proteins. (G,H) Overexpression of dominant-negative mutants that block endocytosis, which neither affected the abundance nor the distribution of the Dvl2 puncta. Bars, 15 µm.

View larger version (137K): [in a new window]   Fig. 5. Dvl2 puncta neither coincide with endocytic vesicles nor stain with lipid dyes. COS-7 cells expressing HA-Dvl2 were fixed and stained with anti-HA antibody (green) after loading of cells with (A-C) fluorescently-labelled conjugates or (D, E) lipid dyes as indicated (red). Bars, 15 µm.

View larger version (15K): [in a new window]   Fig. 6. GFP-Dvl2 puncta can grow by collision and fusion. Series of individual frames (30 seconds apart) taken from supplementary material Movie 2, depicting the fusion of two GFP-Dvl2 puncta in transfected COS-7 cells.

View larger version (59K): [in a new window]   Fig. 7. GFP-Dvl2 puncta do not colocalise with clathrin-DsRed in live COS-7 cells. An individual frame, taken from supplementary material Movie 3 (frame 15), shows distinct (A) GFP-Dvl2 puncta [green in merged image (C)] and (B) clathrin-DsRed puncta in live transfected COS-7 cells.

View larger version (89K): [in a new window]   Fig. 8. Morphology of GFP-Dvl2 puncta at high magnification. (A,B) Single confocal sections of COS-7 cells expressing GFP-Dvl2; (C) GFP-Dvl2 puncta (green in merged image) were also stained with anti-Dvl2 antibody (red in merge); high magnification view in (B) corresponds to boxed section in (A). Notice the multi-centred `puncta' in (B). (D-F) Single confocal sections through individual GFP-Dvl2 puncta (bottom left) and orthogonal reconstructions along the two coloured axes as indicated (above and to the right, respectively) from image stacks. (D,E) deconvoluted images; (F) original image corresponding to (E). All cells were fixed before imaging.

View larger version (45K): [in a new window]   Fig. 9. Dynamic exchange of DIX-domain proteins between punctate and cytosolic pools. Average-sized puncta of (A-C) GFP-Dvl2, (E-G) GFP-DIX or (I-K) GFP-Axin were highlighted with white boxes and bleached, and fluorescence recovery was monitored for the following 3 minutes (at 5-second intervals); unbleached neighbouring puncta (yellow boxes) served as internal controls. (D,H,L) Graphs showing mean fluorescence intensities within the boxes indicated (of bleached puncta, or unbleached controls), were normalised to the mean fluorescence intensity of the whole cell.