JCS02647 Supplementary Material

Files in this Data Supplement:

• Supplemental Table - Adobe PDF
• Supplemental Figure 1 -

  Fig. S1. Schematic of Dvl-2-ER fusion proteins used. The positions of the DIX, PDZ and DEP domains follow published data (Zhang et al., 2000).

• Supplemental Figure 2 -

  Fig. S2. Inducible activation of TCF-dependent transcription by HA-Dvl-2-ER proteins. Activity of HA-Dvl-2-ER mutants in TCF-dependent transcription assay. 293 cells were transiently transfected with reporter plasmids and the indicated constructs. Cultures were re-fed with medium containing 1 mM estradiol or control medium 24 hours before analysis for luciferase activity and protein levels in the detergent-soluble and -insoluble fractions of the cell extracts. Analysis of the cell lysates demonstrated the HA-Dvl-2-ER, the DDIX and the K446M fusion proteins were present in both detergent-soluble and -insoluble extracts from b-estradiol-treated cells at much higher levels than in ethanol-vehicle-treated cells. These results suggested that b-estradiol treatment stabilised the Dvl-2 fusion proteins in both these compartments. In contrast, stabilisation of the DPDZ protein only occurred in the insoluble fraction.

• Supplemental Figure 3 -

  Fig. S3. Serial optical sections of single-copy HA-Dvl-2-ER cells. Cells were treated with 1mM b-estradiol for 1 hour, fixed in 4% paraformaldehyde in PBS and stained for HA-Dvl-2-ER (green) and F-actin (red). Each section (A-I) is an overlaid panel of the staining patterns and represents a 0.5 mm step down through the cell, although the whole cell is not represented. Juxtamembrane HA-Dvl-2-ER staining appeared only where cortical actin staining was also present. Bar, 10 mm.

• Supplemental Figure 4 -

  Fig. S4. Coexpression of V5-Frizzled 4 with HA-Dvl-2-ER. HEK293 cells were either singly transfected with the V5-Frizzled 4 plasmid or the HA-Dvl-2-ER plasmid or co-transfected with both. After 24 hours, transfected cells were treated with ethanol vehicle only or with 1mM b-estradiol for 30 minutes. Cells were then fixed and all were double stained with antibodies to the V5 and HA epitope tags. Note that in the singly transfected controls, there was no cross-reactivity of the antibodies. Also note that b-estradiol treatment had no effect on V5-Frizzled 4 localisation and that in the single transfection the HA-Dvl-2-ER responded to the b-estradiol treatment as previously observed. In contrast, co-transfection resulted in membrane localisation of the HA-Dvl-2-ER in both vehicle and b-estradiol treated cells. Bar, 15 mm.

• Supplemental Figure 5 -

  Fig. S5. Sucrose density gradient flotation experiment. HA-Dvl-2-GFP-transfected CHO cells were analysed as described in the Materials and Methods. Transfection efficiency was estimated at approximately 40%. (A) Western blot analysis of gradient fractions showing transferrin receptor (top) and clathrin heavy chain (middle) localise to the membrane floatation fractions (4 to 6) whereas HA-Dvl-2-GFP (bottom) is found mainly with the non-membrane-associated fraction (12) but also, to a lesser extent, distributed throughout the gradient. (B) Results of densitometry analysis of gradient fractions. The density interface is indicated by the arrows.

• Movie 1A
• Movie 1B
• Movie 1C -

  Movie 1. (A) Time-lapse analysis of HA-Dvl-2-EGFP expression following microinjection into MDCK cells. (B) Twofold magnified time-lapse analysis of a cell expressing HA-Dvl-2-EGFP at high levels in upper right field of Movie 1A. (C) Twofold magnified time-lapse analysis of a cell expressing HA-Dvl-2-EGFP at lower levels in lower left field of Movie 1A.

• Movie 2A
• Movie 2B -

  Movie 2. (A) Improvision Volocity 3DM time-lapse analysis of HA-Dvl-2-EGFP transient ectopic expression in HEK293 cells. (B) Improvision Volocity 3DM time-lapse analysis of HA-Dvl-2-EGFP transient ectopic expression in HEK293 cells. Three-dimensional still reconstruction of data gathered in Movie 2A.