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First published online July 2, 2007
doi: 10.1242/10.1242/jcs.002956

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Dynamic recruitment of axin by Dishevelled protein assemblies

Thomas Schwarz-Romond^*, Ciara Metcalfe and Mariann Bienz

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

View larger version (14K): [in this window] [in a new window]   Fig. 1. The domain structures of Dvl and axin proteins. Schematic representations of Dvl2 and axin, with their ligands binding to their known structural domains PDZ (Wong et al., 2003), DEP (Wong et al., 2000) and RGS (Spink et al., 2000); the DIX and DAX domains mediate dynamic polymerisation (Schwarz-Romond et al., 2007); the sequence stretches in axin that bind to -catenin and GSK3 are also indicated (in black); residue numbers underneath domains denote the domain boundaries. Note that the main binding domain for CKI appears to be the DEP domain (Kishida et al., 2001) (see below, Fig. 6), but this kinase could also bind to the PDZ domain (Peters et al., 1999).

View larger version (84K): [in this window] [in a new window]   Fig. 2. Wnt-dependent relocation of Dvl2-GFP and endogenous Dvl2 puncta to the plasma membrane. (A,B) Fixed 293T cells, after transfection with Dvl2-GFP (50 ng), Fz8 (100 ng), LRP6 (100 ng), GSK3 (100 ng) and HA-axin (100 ng), (A) before or (B) after 2 hours of exposure to recombinant Wnt3a, showing Wnt-dependent recruitment of Dvl2-GFP to the plasma membrane. (C-F) 293T cells, (C-E) before or (F) after exposure to Wnt3a as in B, fixed and stained with affinity-purified anti-Dvl2 antiserum, revealing endogenous cytoplasmic Dvl2 puncta (C) that are recruited to the plasma membrane (F, arrows) in a Wnt-dependent way (insert in C, high-magnification view of cytoplasmic puncta of endogenous Dvl2); the specificity of the signals is indicated by the near-complete elimination of the staining after (D) siRNA treatment against Dvl2, or (E) prior depletion of the antiserum with 100x molar excess of recombinant GST-DIX. Scale bar, 15 µm.

View larger version (39K): [in this window] [in a new window]   Fig. 3. The DAX domain is required for efficient puncta formation. (A-C) COS-7 cells expressing wt or mutant axin, as indicated were fixed, and (C) stained with antibody against HA (the punctate pattern of wt HA-axin is essentially the same as in A; not shown). Note that DAX and HA-axin_M12 (a mutant version of axin, the self-association of which is expected to be blocked; see text) produce a less punctate, more diffuse pattern, indicating that the ability of axin to form puncta depends at least partly on its DAX domain. Scale bar, 15 µm.

View larger version (41K): [in this window] [in a new window]   Fig. 4. The recruitment of axin into Dvl2 puncta depends on the DIX but not the DAX domain. (A-E) COS-7 cells co-expressing wt and mutant axin (left-hand panels, and red) and Dvl2 (middle panels, and green) as indicated (merges in right-hand panels), fixed and stained with antibodies against Dvl2 and (A,E) HA. Axin colocalises precisely with Dvl2 puncta (A), regardless of its DAX domain (B), but dependent on a functional DIX domain of Dvl2 (C,D; the DIX construct used here also contains the TPR dimerisation domain, but this domain does not detectably change its behaviour or subcellular distribution; not shown). Note that two sets of puncta are observed if the DIX domain is co-expressed with axin, indicating that the DIX and DAX domains do not interact directly. (F) Pull-down assays between bacterially expressed GST-tagged DIX domain and in vitro translated HA-tagged axin or Dvl2, as indicated (GST input proteins are shown on the right). The DIX domain binds efficiently to itself (within full-length HA-Dvl2, arrow), but not to the DAX domain (within full-length HA-axin, arrow). Scale bar, 15 µm.

View larger version (21K): [in this window] [in a new window]   Fig. 5. The recruitment of axin into Dvl2 puncta requires extensive DIX-domain flanking sequences. (A) TCF transcription assays in 293 cells, transfected with a TCF-dependent firefly luciferase reporter (SuperTOP; 50 ng) and an internal control (CMV-renilla; 25 ng), and with full-length Dvl2 (200 ng) or Dvl2 truncations as indicated (500 ng); bars indicate standard deviations. (B) Western blot, probed with anti-FLAG antibody, showing expression levels of Flag-Dvl2 constructs, as indicated. (C-F) HeLa cells, cotransfected with HA-axin (green) and full-length FLAG-Dvl2 or truncations (red), as indicated in panels, fixed and stained with antibodies against their tags; note the two sets of cytoplasmic puncta in D (red or green), but the co-localisation of Dvl and axin puncta in F (yellow), indicating that the minimal axin-recruiting sequences of Dvl2 are contained within amino acids 1-389. Scale bar, 15 µm.

View larger version (16K): [in this window] [in a new window]   Fig. 6. Recruitment of CKI into Dvl2 puncta. (A) SuperTOP assays in transfected 293 cells as in Fig. 5A; limiting amounts of HA-CKI (100 ng) were co-expressed with full-length FLAG-Dvl2 (200 ng) or truncations (500 ng), as indicated. (B-E) HeLa cells, cotransfected with HA-CKI (green) and FLAG-Dvl2 or truncations (red) as indicated in panels, fixed and stained as in Fig. 5. Note that Dvl299 does not affect the HA-CKI staining, which remains diffuse cytoplasmic (D), indistinguishable from the staining of HA-CKI expressed by itself, or co-expressed with DIX (not shown). (F) SuperTOP assays in 293T cells transfected with FLAG-Dvl2, after RNAi-mediated depletion of endogenous CKI (to <50% of normal levels, as show by western blotting, G), revealing that the signalling activity of Dvl2 depends on CKI. Scale bar, 15 µm.

View larger version (21K): [in this window] [in a new window]   Fig. 7. Dvl2 accelerates the recruitment of axin into dynamic protein assemblies. (A,B) Graphs of FRAP experiments conducted in COS-7 cells, in which individual (A) axin-GFP or (B) Dvl2-GFP puncta were bleached, and fluorescence recovery within the bleached boxes was monitored every 5 seconds (coloured lines represent different puncta; values were normalised with respect to unbleached control puncta, shown by dotted line). (C-G) Graphs of FRAP experiments of axin-GFP as in A, but in the presence of co-expressed wt or mutant Dvl2, as indicated. Note that wt but not mutant Dvl2 accelerates the recovery rates of axin-GFP.

View larger version (23K): [in this window] [in a new window]   Fig. 8. Dvl2-axin puncta remain dynamic after Wnt-induced recruitment to the plasma membrane. (A-D) Fixed HeLa cells, co-expressing Fz8, LRP6 and GSK3 (as in Fig. 1B) in addition to (A,B) HA-Dvl2 plus axin-GFP or (C,D) Dvl2-GFP plus HA-axin, (A,C) before or (B,D) after 2 hours of exposure to recombinant Wnt3a. (E,F) Graphs of FRAP experiments conducted in HeLa cells, transfected and Wnt-stimulated as in B,D; individual membrane-associated puncta were bleached, and fluorescence recovery of (E) axin-GFP or (F) Dvl2-GFP was monitored, as in Fig. 7. Note that the recovery rates of these puncta are similar to those of cytoplasmic Dvl2-GFP puncta, and of axin-GFP recruited into cytoplasmic Dvl2 puncta, in HeLa cells (not shown) and in COS-7 cells (Fig. 7B-D).