Fagotto F et al. (1996), Binding to cadherins antagonizes the signaling ...

XB-ART-18506

J Cell Biol 1996 Mar 01;1326:1105-14. doi: 10.1083/jcb.132.6.1105.

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Binding to cadherins antagonizes the signaling activity of beta-catenin during axis formation in Xenopus.

Fagotto F , Funayama N , Gluck U , Gumbiner BM .

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beta-Catenin, a cytoplasmic protein known for its association with cadherin cell adhesion molecules, is also part of a signaling cascade involved in embryonic patterning processes such as the determination of the dorsoventral axis in Xenopus and determination of segment polarity in Drosophila. Previous studies suggest that increased cytoplasmic levels of beta-catenin correlate with signaling, raising questions about the need for in- teraction with cadherins in this process. We have tested the role of the beta-catenin-cadherin interaction in axis formation. Using beta-catenin deletion mutants, we demonstrate that significant binding to cadherins can be eliminated without affecting the signaling activity. Also, depletion of the soluble, cytosolic pool of beta-catenin by binding to overexpressed C-cadherin completely inhibited beta-catenin-inducing activity. We conclude that binding to cadherins is not required for beta-catenin signaling, and therefore the signaling function of beta-catenin is independent of its role in cell adhesion. Moreover, because beta-catenin signaling is antagonized by binding to cadherins, we suggest that cadherins can act as regulators of the intracellular beta-catenin signaling pathway.

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Species referenced: Xenopus laevis
Genes referenced: cdh3 mt4 mtnr1a myc

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	Figure 1. Synopsis of the 13-catenin mutants used in this work. Prefix 13, untagged; prefix HT, HA-tagged; and prefix MT, myctagged. 13-Catenin sequence contains a gap (white case) between repeats 10 and 11. See Materials and Methods for the~precise coding sequence of each construct.
	Figure 2. Assay for 13-catenin signaling: axis duplication in Xenopus embryos. (A) Example of tadpole with two complete body axes, including two heads, obtained by injection of 0.75 ng of a truncated 13-catenin mutant mRNA, MT10, in the ventral side of a cleaving embryo. (B) Normal tadpole developed from an uninjected embryo.
	Figure 3. Association of mutants HT6-HT9 with endogenous C-cadherin and APC. (a) Expression of full-length 13-catenin (HT1) and mutants HT6-HT9 in Xenopus embryos injected with 3 ng mRNA of each construct. The mutants were detected with a mixture of two pAb raised against the NH2 and COOH terminus of Xenopus 13-catenin, respectively. (b) Detection of C-cadherin in [3-catenin mutant immunoprecipitates under mild conditions (nonionic detergent). Mutants were immunoprecipitated with anti-HA mAb 12CA5 in the presence of 1% NP-40. C-cadherin is associated with HT1, HT8 and HT9, but not HT6 and HT7. (c) Detection of C-cadherin in [3-catenin mutant immunoprecipitates under harsher conditions (anionic detergent). Mutants were immunoprecipitated with anti-HA mAb 12CA5 in the presence of RIPA buffer, containing 0.2% SDS, 1% Triton X-100 and 0.5% deoxycholate. C-cadherin is associated with HT1 and HT9, but not HT6, HT7, nor HT8. (d) Detection of APC in 13-catenin mutant immunoprecipitates. Mutants were immunoprecipitated with the anti-HA mAb 12CA5 in the presence of NP-40. C-cadherin is associated with HT1, HT8 and HT9, but not HT6 and HT7.
	Figure 4. Lack of association between MT10 and C-cadherin. (a) Expression of full-length 13-catenin (MT1) and MT10, containing only the first nine armadillo repeats, in Xenopus embryos injected in the two ventral blastomeres of a 4-cell stage embryo with 2 x 3 ng mRNA. MT1 and MT10 were detected with the anti-myc tag mAb 9E10.2. (b) Detection of MT1 and MT10 after immunoprecipitation with anti-myc mAb 9E10.2 in the presence of 1% NP-40. (c) Detection of C-cadherin in MT1 and MT10 immunoprecipitates (1% NP-40). The first lane shows a control immunoprecipitation from uninjected embryos. C-cadherin (arrow) is associated only with full-length 13-catenin, but not MT10.
	Figure 5. Ventralization of Xenopus embryos by overexpression of C-cadherin in the dorsal inducing region. (A) When two dorsal blastomeres of 4-cell stage embryos were injected at the vegetal pole with 4--5 ng C-cadherin mRNA, the embryos developed into ventralized tadpoles (no head, diminished dorsal structures, hypertrophy of ventral tissues). (B) After injections of the same RNA in the ventral side, the embryos developed normally.
	Figure 6. Inhibition of 13-catenin-indueed axis duplication by coexpression of C-cadherin. (A and C) Embryos injected with 0.3 ng full-length I~-catenin (HT1) mRNA developed duplicated axis, visible at neurula stage by the two pigmented neural tubes (,4) and at tadpole stage as two complete body axis, including two heads. (B and D) Embryos coinjected with 0.3 ng HT1 and 5 ng C-cadherin mRNA have only one axis (B) and develop into normal tadpoles (D).
	Figure 7. Redistribution of fulllength ~3-catenin (MT1) but not MT10 by coexpression of C-cadherin. Full-length 13-catenin (MT1) or MT10 (both 1.5 ng mRNA), expressed alone (A and C) or coexpressed with excess C-cadherin (5 ng mRNA) (B and D) were immunolocalized at early gastrula stages by indirect immunofluorescence on frozen sections with anti-myc mAb 9E10.2. (A '-D') HOECHST staining to visualize nuclei on the same sections. Cell-cell boundaries are outlined with dotted lines in panels C' and D '. (A) Full-length MT1 expressed alone is detected in the cytoplasm (i.e., between the yolk platelets, which appear as large dark granules), and accumulates at the plasma membrane and in the nuclei (arrowheads). (B) In the presence of excess C-cadherin, MT1 is completely redistributed to the plasma membrane and intracellular vesicles (arrows). Cytoplasm and nuclei (arrowheads) are depleted. (C) The mutant MT10 expressed alone is localized in the cytoplasm and the nuclei (arrowheads), but not at the membrane. The cell periphery often appears brighter because it contains less yolk. The peripheral cytoplasmic staining closely matches the irregular outline of the yolk platelets (small arrows) and can be unambiguously distinguished from the sharper, straight line of membrane staining observed for full-length 13-catenin (A and B), endogenous 13-catenin and C-cadherin (not shown, see Fagotto and Gumbiner, 1994). (D) MT10 coexpressed with C-cadherin is distributed throughout the cytoplasm, with no enrichment at the cell membrane. Some punctate intracellular staining is observed (arrow). Arrowheads point to nuclei. Bar, 20 t~m.
	Figure 8. Redistribution of full-length 13-catenin (MT1) but not MT10 by coexpression of C-cadherin: cell fractionation. Late blastula embryos expressing MT1 alone (a), MT1 with excess C-cadherin (b), MT10 alone (c), and MT10 with excess C-cadherin (d) were fractionated as described in Materials and Methods into a membrane glycoprotein (cadherins) fraction (G); a particulate noncadherin-bound fraction (P), and a soluble fraction (S). MT1 and MT10 were detected on Western blots with anti-myc mAb 9E10.2.

References [+] :

Amaya, Expression of a dominant negative mutant of the FGF receptor disrupts mesoderm formation in Xenopus embryos. 1991, Pubmed, Xenbase