RESUMO
The activation of distinct branches of the Wnt signaling network is essential for regulating early vertebrate development. Activation of the canonical Wnt/ß-catenin pathway stimulates expression of ß-catenin-Lef/Tcf regulated Wnt target genes and a regulatory network giving rise to the formation of the Spemann organizer. Non-canonical pathways, by contrast, mainly regulate cell polarization and migration, in particular convergent extension movements of the trunk mesoderm during gastrulation. By transcriptome analyses, we found caveolin1, caveolin3 and cavin1 to be regulated by Lef1 in the involuting mesoderm of Xenopus embryos at gastrula stages. We show that caveolins and caveolin dependent endocytosis are necessary for proper gastrulation, most likely by interfering with Wnt5a/Ror2 signaling. Wnt5a regulates the subcellular localization of receptor complexes, including Ror2 homodimers, Ror2/Fzd7 and Ror2/dsh heterodimers in an endocytosis dependent manner. Live-cell imaging revealed endocytosis of Ror2/caveolin1 complexes. In Xenopus explants, in the presence of Wnt5a, these receptor clusters remain stable exclusively at the basolateral side, suggesting that endocytosis of non-canonical Wnt/receptor complexes preferentially takes place at the apical membrane. In support of this blocking endocytosis with inhibitors prevents the effects of Wnt5a. Thus, target genes of Lef1 interfere with Wnt5a/Ror2 signaling to coordinate gastrulation movements.
Assuntos
Caveolina 3/metabolismo , Endocitose , Regulação da Expressão Gênica no Desenvolvimento , Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriologia , Animais , Caveolina 1/genética , Caveolina 1/metabolismo , Caveolina 3/genética , Feminino , Gastrulação , Fator 1 de Ligação ao Facilitador Linfoide/genética , Masculino , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/genética , Transdução de Sinais , Proteína Wnt-5a/genética , Proteína Wnt-5a/metabolismo , Proteínas de Xenopus/genética , Xenopus laevis/genética , Xenopus laevis/metabolismoRESUMO
Diatoms are unicellular organisms evolved by secondary endosymbiosis. Although studied in many aspects, the functions of vacuolar-like structures of these organisms are rarely investigated. One of these structures is a dominant central vacuole-like compartment with a marbled phenotype, which is supposed to represent a chrysolaminarin-storing and carbohydrate mobilization compartment. However, other functions as well as targeting of proteins to this compartment are not shown experimentally. In order to study trafficking of membrane proteins to the vacuolar membrane, we scanned the genome for intrinsic vacuolar membrane proteins and used one representative for targeting studies. Our work led to the identification of several proteins located in the vacuolar membrane as well as the sub-compartmentalized localization of one protein. In addition, we show that a di-leucine-based motif is an important signal for correct targeting to the central vacuole of diatoms, like it is in plants.
