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      In vivo analysis of formation and endocytosis of the Wnt/β-Catenin signaling complex in zebrafish embryos

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          ABSTRACT

          After activation by Wnt/β-Catenin ligands, a multi-protein complex assembles at the clustering membrane-bound receptors and intracellular signal transducers into the so-called Lrp6-signalosome. However, the mechanism of signalosome formation and dissolution is yet not clear. Our imaging studies of live zebrafish embryos show that the signalosome is a highly dynamic structure. It is continuously assembled by Dvl2-mediated recruitment of the transducer complex to the activated receptors and partially disassembled by endocytosis. We find that, after internalization, the ligand-receptor complex and the transducer complex take separate routes. The Wnt–Fz–Lrp6 complex follows a Rab-positive endocytic path. However, when still bound to the transducer complex, Dvl2 forms intracellular aggregates. We show that this endocytic process is not only essential for ligand-receptor internalization but also for signaling. The μ2-subunit of the endocytic Clathrin adaptor Ap2 interacts with Dvl2 to maintain its stability during endocytosis. Blockage of Ap2μ2 function leads to Dvl2 degradation, inhibiton of signalosome formation at the plasma membrane and, consequently, reduction of signaling. We conclude that Ap2μ2-mediated endocytosis is important to maintain Wnt/β-catenin signaling in vertebrates.

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          Proximal events in Wnt signal transduction.

          The Wnt family of secreted ligands act through many receptors to stimulate distinct intracellular signalling pathways in embryonic development, in adults and in disease processes. Binding of Wnt to the Frizzled family of receptors and to low density lipoprotein receptor-related protein 5 (LRP5) or LRP6 co-receptors stimulates the intracellular Wnt-beta-catenin signalling pathway, which regulates beta-cateninstability and context-dependent transcription. This signalling pathway controls many processes, such as cell fate determination, cell proliferation and self-renewal of stem and progenitor cells. Intriguingly, the transmembrane receptor Tyr kinases Ror2 and Ryk, as well as Frizzledreceptors that act independently of LRP5 or LRP6, function as receptors for Wnt and activate beta-catenin-independent pathways. This leads to changes in cell movement and polarity and to the antagonism of the beta-catenin pathway.
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            Dishevelled: The hub of Wnt signaling.

            Wnt signaling controls a variety of developmental and homeostatic events. As a key component of Wnt signaling, Dishevelled (Dvl/Dsh) protein relays Wnt signals from receptors to downstream effectors. In the canonical Wnt pathway that depends on the nuclear translocation of beta-catenin, Dvl is recruited by the receptor Frizzled and prevents the constitutive destruction of cytosolic beta-catenin. In the non-canonical Wnt pathways such as Wnt-Frizzled/PCP (planar cell polarity) signaling, Dvl signals via the Daam1-RhoA axis and the Rac1 axis. In addition, Dvl plays important roles in Wnt-GSK3beta-microtubule signaling, Wnt-calcium signaling, Wnt-RYK signaling, Wnt-atypical PKC signaling, etc. Dvl also functions to mediate receptor endocytosis. To fulfill its multifaceted functions, it is not surprising that Dvl associates with various kinds of proteins. Its activity is also modulated dynamically by phosphorylation, ubiquitination and degradation. In this review, we summarize the current understanding of Dvl functions in Wnt signal transduction and its biological functions in mouse development, and also discuss the molecular mechanisms of its actions. 2009 Elsevier Inc. All rights reserved.
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              Wnt induces LRP6 signalosomes and promotes dishevelled-dependent LRP6 phosphorylation.

              Multiple signaling pathways, including Wnt signaling, participate in animal development, stem cell biology, and human cancer. Although many components of the Wnt pathway have been identified, unresolved questions remain as to the mechanism by which Wnt binding to its receptors Frizzled and Low-density lipoprotein receptor-related protein 6 (LRP6) triggers downstream signaling events. With live imaging of vertebrate cells, we show that Wnt treatment quickly induces plasma membrane-associated LRP6 aggregates. LRP6 aggregates are phosphorylated and can be detergent-solubilized as ribosome-sized multiprotein complexes. Phospho-LRP6 aggregates contain Wnt-pathway components but no common vesicular traffic markers except caveolin. The scaffold protein Dishevelled (Dvl) is required for LRP6 phosphorylation and aggregation. We propose that Wnts induce coclustering of receptors and Dvl in LRP6-signalosomes, which in turn triggers LRP6 phosphorylation to promote Axin recruitment and beta-catenin stabilization.
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                Author and article information

                Journal
                J Cell Sci
                J. Cell. Sci
                joces
                jcs
                Journal of Cell Science
                The Company of Biologists (Bidder Building, 140 Cowley Road, Cambridge, CB4 0DL, UK )
                0021-9533
                1477-9137
                15 September 2014
                15 September 2014
                : 127
                : 18
                : 3970-3982
                Affiliations
                [1 ]Karlsruhe Institute of Technology (KIT), Institute of Toxicology and Genetics (ITG) , 76021 Karsruhe, Germany
                [2 ]Departments of Cell Biology and Pediatrics, Harvard Medical School and Program in Cellular and Molecular Medicine at Boston Children's Hospital , Boston, 02115 MA, USA
                Author notes
                [*]

                Present address: Broad CIRM Center, University of Southern California, Keck School of Medicine, Los Angeles, 90089 CA, USA.

                []Author for correspondence ( steffen.scholpp@ 123456kit.edu )
                Article
                10.1242/jcs.148767
                4163645
                25074807
                596fe88e-a04f-40e7-a3e8-5461e3324e77
                © 2014. Published by The Company of Biologists Ltd

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

                History
                : 31 December 2013
                : 7 July 2014
                Categories
                Research Article
                124

                Cell biology
                wnt signaling,development,endocytosis,signalosome,zebrafish
                Cell biology
                wnt signaling, development, endocytosis, signalosome, zebrafish

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