Fagotto F , Jho Eh , Zeng L , Kurth T , Joos T , Kaufmann C , Costantini F .

GM56934 NIGMS NIH HHS

	Figure 8. Cell fractionation of Myc–Axin in Xenopus embryos: sedimentability and Con A binding. (A) Diagram of Axin molecule. (B) Myc–Axin is associated with a sedimentable fraction. Homogenates of late blastula embryos were fractionated into a low speed sedimentable fraction (P, pellet), a high speed sedimentable fraction (M, membranes), and a high speed supernatant (S, soluble fraction) as described in Materials and Methods. β-Galactosidase was coexpressed and used as a control for soluble cytosolic proteins. Unlike β-galactosidase, Myc–Axin was sedimentable under these conditions. (C) Myc–Axin is fully extractable in NP-40. Embryos expressing Myc–Axin were extracted in NP-40–containing buffer (sol). The insoluble pellet was reextracted in the presence of SDS (insol, NP-40– insoluble). (D) A pool of Axin is associated with a membrane glycoprotein, and this association requires the NH2-terminal domain. Myc-tagged FL Axin and various Axin mutant constructs were expressed in embryos, and NP-40 extracts were fractionated using Con A beads. Bound fractions (B) were four times concentrated relative to unbound fractions (U). FL Axin showed significant association with Con A beads that indicates a stable interaction with a membrane glycoprotein. Stronger binding was observed for the NH2-terminal fragments Ax12-531. On the other hand, constructs lacking the NH2-terminal domain showed no (Ax-531-956 and Ax194-353) or very weak binding (Ax194-956).
	Figure 1. In vivo interaction of Myc-tagged Axin and mutant Axin constructs with endogenous β-catenin (A) or GSK3β (B). Myc-tagged Axin constructs were expressed in 293 cells. Cell lysates were immunoprecipitated with anti-Myc mAb and immunoprecipitates were analyzed for endogenous β-catenin or GSK3β by Western blot (upper panels in A and B). Expression of the transfected Myc– Axin constructs (asterisks) is shown in the lower panels.
	Figure 2. Axin binds to APC and induces APC phosphorylation in vivo. (A) Diagram showing the regions of human APC included in three APC constructs. (B) Direct in vitro binding of APC25 to Axin fragments containing the RGS domain. Sulfur-35–labeled APC25 protein was incubated with S-tagged fusion proteins containing the indicated regions of Axin. After S-protein agarose IP, bound APC25 was detected by SDS-PAGE and autoradiography. Coomassie blue staining of the Axin fusion proteins is shown at the bottom. (C) In vitro binding with NH2-terminal Axin fragments reveals a second APC-binding site upstream of the RGS domain. Binding of APC2 and APC25 required the RGS domain (Ax96-353 and Ax96-480). However, FL hAPC also interacted with Ax96-253, and hAPC2 bound Ax96-253 very strongly. Interaction of Sulfur-35–labeled FL APC, APC2, APC21 and APC25 with S-tagged fusion proteins was determined as in B. (D) CoIP of VSV–APC with FL Axin and several mutant forms of Axin. 293 cells were cotransfected with VSV-G–tagged Xenopus APC (VSV–APC) and the indicated Axin constructs. The cell lysates were immunoprecipitated with anti-Myc and probed with anti–VSV-G antibody. Failure to detect interaction with Ax12-355 was due to the very low levels of VSV–APC observed when coexpressed with this particular construct (E). (E and F) Axin-dependent phosphorylation of APC. (E) Axin-induced mobility shift of VSV–APC. Total lysates from cotransfected cells were analyzed by Western blot using an anti-VSV mAb. FL Axin and mutants Ax12-810, 12-600, and 12-531 induced a mobility shift, whereas AxΔ251-351, Ax12-355, and Ax497-622 did not. (F) Phosphatase treatment eliminated the mobility shift. Cotransfected cell lysates were IP with anti-VSV mAb, and the products were analyzed by SDS-PAGE and Western blot before (left) or after (right) incubation with λ-protein phosphatase.
	Figure 3. Summary of properties of FL and mutant forms of Myc-tagged Axin. The diagram at the top indicates homology domains and binding sites for other proteins based on data reported here and previously (see text for references). Numbers at left indicate the NH2- and COOH-termini, and any internal deletion (Δ). Dotted lines indicate deletions. Vent., extent of ventralization (+++ or ++) or dorsalization (D, strong or d, weaker, and only at high concentration) upon injection into Xenopus embryos. β-cat level indicates the effect on the level of expression of coinjected HA-tagged β-catenin: up arrow, increase; side arrow, no change; and down arrow, decrease. coIP indicates coimmunoprecipitation with the indicated protein (VSV–APC, endogenous GSK3β, or endogenous β-catenin) after transient transfection into 293 cells. +/− indicates that coIP was weak and sporadic in multiple assays. The question mark indicates that the coIP of APC could not be determined because expression of these two Axin mutants greatly reduced the total level of cellular APC (E and data not shown). Localiz. indicates a pattern of intracellular localization after injection into Xenopus embryos. Sp and sp, strong or weak localization to spots, respectively; M and m, strong or weak plasma membrane staining, respectively; df, diffuse cytoplasmic staining; asterisk, pattern was slightly particulate, in contrast to other mutants labeled df. A blank space indicates not determined.
	Figure 4. Ventralization and dorsalization by expression of Axin mutants in Xenopus embryos. (A) Diagram of Axin molecule. The main binding domains are indicated as well as the aa numbers corresponding to most restriction sites used in the generation of mutant Axin constructs. (B) Examples of ventralized embryos (tailbud stage) obtained by dorsal injection of Ax194-672 mRNA. Two embryos have greatly reduced axes (arrowheads) and no head. The other four embryos are completely ventralized (i.e., they lack dorsal structures and have no body axis). (C) Embryos expressing Ax531-956 develop normally. (D) Examples of hyperdorsalized embryos obtained by dorsal expression of Ax331-956. These embryos have enlarged heads and cement glands (large arrows) or double cement glands (small arrows). (E) Partially dorsalized embryos resulting from dorsal expression of Ax194-531. These embryos typically form very large semicircular cement glands (arrows) and their heads are generally strongly disorganized (arrowheads). (F) Axis duplication by ventral expression of Ax331-956. Most of the embryos form complete twin embryos, including two cement glands (arrows). (G) Expression levels of FL and mutant Axin constructs in embryos. (Left) An example of a series of constructs expressed dorsally. (Right) Expression levels of a series of constructs with internal deletion of the RGS domain, expressed ventrally. Note that 1–2 ng Ax12-956Δ251-351 mRNA induced complete duplicated axes (see Table II), whereas the other constructs expressed at similar or even higher levels induced mostly partial or no secondary axis.
	Figure 6. Myc-tagged Axin localizes in discrete spots and colocalizes with HA–Dsh. (A) Cellular distribution of FL Myc–Axin (green, Alexa488 staining) in Xenopus blastulae, detected by indirect IF on frozen sections. Myc–Axin localized mainly in spots (small arrows) or clusters of spots (large arrows), found generally but not exclusively at the cell periphery. Some plasma membrane staining was also observed (arrowhead). Red shows yolk platelets counterstained with Eriochrome Black; blue shows the nuclei (DAPI staining). (A′) Large cluster of Myc–Axin-positive spots (arrows) at higher magnification. (A′′) Detail of membrane localization of Myc–Axin (arrowheads). Spots also are found at or near the cell surface (arrow). (B and B′) Localization of FL Myc–Axin in transfected HeLa cells. Myc–Axin (red, Cy3 staining) is found in spots, mostly at the cell periphery. In high expressing cells, bright cytoplasmic clusters were observed (B′, arrows). (B′′) Detail of two clusters at high magnification (arrows). Note that images in A and A′′ and B and B′′ were collected under very different conditions (exposure, filters), since the signal was one order of magnitude stronger in transfected HeLa cells compared with mRNA-injected embryos. Blue shows the nuclei (DAPI staining). (C and C′) Colocalization of FL Myc–Axin and HA–Dsh. Coexpressed Myc–Axin (green, Alexa488 staining) and HA–Dsh (red, Cy3 staining) were detected on sections from Xenopus blastulae by double IF. All Myc–Axin colocalized perfectly with HA–Dsh (arrowheads), but some HA–Dsh spots did not stain for Myc–Axin (arrows). Note that most Axin-Dsh spots are distributed along the periphery of the cells. (D) Localization of HA–Dsh expressed alone: Dsh (red, Cy3 staining) localizes in spots, distributed throughout the cytoplasm. Blue shows nuclei (DAPI). Bars: (A) 10 μm; (A′) 2 μm; (A′′) 5 μm; (B and B′) 10 μm; (B′′) 5 μm; (C and D) 20 μm.
	Figure 7. Electron microscopic localization of Myc– Axin. (A–C) EM localization of FL Myc–Axin by on-section staining on Lowycryl sections. (A) Low magnification view of an Axin-positive cluster detected by indirect IF on a thin section. Axin expression in this cell is relatively low and individual spots can be resolved (arrows). (B) Low magnification EM image of a cluster of gold particles (15 nm) labeling an area of dense cytoplasm (arrows) containing numerous vesicles (asterisks). Note that outside the cluster the surrounding cytoplasm is devoid of gold particles. (C) High magnification view of portion of a less dense cluster. The cluster is composed of small groups of gold particles decorating electron dense cytoplasm (arrows) associated with a few vesicles (asterisks). Each small group probably corresponds to a single “spot” observed by IF (A and Fig. 6, A and B). Note that the membranes surrounding the vesicular structures appear much less contrasted in B and C compared with E. This is due to the difference in the methods used (low contrast Lowycryl sections in B and high contrast conventional Spurr sections in D). (D and E) EM localization of Myc–Axin by preembedding Nanogold labeling and silver enhancement. (D) Low magnification view of a Myc–Axin positive area (arrow) in a high expressing cell. Single gold aggregates found both in the cytoplasm and in the nucleus (n) represent background that is higher with this preembedding technique. The arrowhead points to the plasma membrane that shows no significant staining in this cell. (E) High magnification view of a similar area, packed with vesicles of variable size (∼50-200 nm) embedded in electron dense cytoplasm. The irregular shape of the gold/silver particles is due to the silver enhancement method. Insert shows enlarged view (2×) of the outlined area, with tightly-packed vesicles (asterisks) and electron dense cytoplasm (arrow). Note that gold labeling tends to be somewhat excluded from the areas particularly packed with vesicles. This could be due to limited diffusion of Nanogold in these preparations. (F) Nanogold localization of Myc–Axin at the plasma membrane. Because FL Axin localization at the membrane is weak (D, and Fig. 7 A), a mutant Axin, AxΔ531-810 was used in this experiment. In this case, the plasma membrane is heavily decorated with gold/silver particles (arrowheads). m, mitochondrion; p, pigment granules; and yp, yolk platelets. Bars: (A) 2 μm; (B) 1 mm; (C) 0.2 μm; (D) 1 μm; (E) 0.5 μm; (F) 1 μm.
	Figure 9. Sequence specific patterns of cellular distribution of Axin mutants. FL and various mutant forms of Myc-tagged Axin were expressed in Xenopus embryos and localized by IF (green) on frozen sections. Diagram of Axin molecule. (B and C) FL and AxΔ251-351 localized preferentially to spots or clusters of spots (arrows). Variable membrane staining was detected in some cells (B and insert C, arrowheads). This pattern was found for all constructs containing both NH2- and COOH-termini. (D) Diffuse cytoplasmic distribution of Ax497-672 found in most constructs lacking the NH2 terminus. (E) Example of a mutant (Ax194-956) showing a cytoplasmic, but somewhat particulate, distribution with some weak enrichment at the cell periphery (arrowhead). Partial cell surface enrichment is characteristic of mutants lacking the NH2 terminus but with intact APC and GSK3β-binding sites. (F and G) Constructs containing the NH2 terminus but lacking the COOH terminus, such as Ax12-672 and Ax12-531, are mainly localized at the plasma membrane (arrowheads). Some intracellular punctate staining is also observed (arrows). (H) Localization of AxΔ352-631 mimics the punctate staining of FL Axin (arrows). (I and J) High magnification views of punctate staining (I, AxΔ352-631) and membrane staining (J, Ax12-355). Bars: (A–G) 20 μm; (H, I, and insert B) 5 μm.
	Figure 10. Axin domains, molecular interactions, and their functions. Binding domains are represented by boxes and direct molecular interactions are indicated by thick lines. Axin, APC, and β-catenin can bind to each other and form a triangular complex. A secondary Axin–APC interaction is shown by a dashed line. GSK3β binding to Axin is required for phosphorylation (arrows and P) of Axin itself, as well as APC, and β-catenin. Phosphorylation of APC regulates APC-β-catenin binding and phosphorylation of β-catenin is required for its degradation. The function of Axin phosphorylation is not known. The catalytic subunit of PP2A binds to a site adjacent to the β-catenin binding site and may counteract the activity of GSK3β (dotted lines with arrows). The COOH terminus contains a dimerization domain, whose function is unclear. Binding to Dsh (dotted line) is suggested by the presence of a DIX domain in both molecules and by colocalization with Axin. Localization of Axin depends on its NH2-terminal and COOH-terminal sequences. The NH2 terminus is required for both plasma membrane and cytoplasmic spot localization, whereas the COOH terminus enhances localization at spots. The RGS domain and GSK3β binding site are required for ventralizing activity. A segment of Axin including the RGS, GSK3β and β-catenin binding domains is sufficient for activity. However, the NH2 terminus can substitute for the β-catenin binding domain, possibly by stabilizing an Axin•APC•β-catenin complex in the absence of direct Axin–β-catenin binding.

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