Activation of the Wnt/β-catenin pathway crucially depends on the polymerization of dishevelled 2 (DVL2) into biomolecular condensates. However, given the low affinity of known DVL2 self-interaction sites and its low cellular concentration, it is unclear how polymers can form. Here, we detect oligomeric DVL2 complexes at endogenous protein levels in human cell lines, using a biochemical ultracentrifugation assay. We identify a low-complexity region (LCR4) in the C-terminus whose deletion and fusion decreased and increased the complexes, respectively. Notably, LCR4-induced complexes correlated with the formation of microscopically visible multimeric condensates. Adjacent to LCR4, we mapped a conserved domain (CD2) promoting condensates only. Molecularly, LCR4 and CD2 mediated DVL2 self-interaction via aggregating residues and phenylalanine stickers, respectively. Point mutations inactivating these interaction sites impaired Wnt pathway activation by DVL2. Our study discovers DVL2 complexes with functional importance for Wnt/β-catenin signaling. Moreover, we provide evidence that DVL2 condensates form in two steps by pre-oligomerization via high-affinity interaction sites, such as LCR4, and subsequent condensation via low-affinity interaction sites, such as CD2.

• Keywords
• DVL2, Wnt signaling, biochemistry, biomolecular condensates, cell biology, chemical biology, dishevelled, human, paralogs,
• MeSH
• beta Catenin metabolism   genetics   MeSH
• HEK293 Cells MeSH
• Humans MeSH
• Protein Multimerization MeSH
• Dishevelled Proteins * metabolism   genetics   MeSH
• Wnt Signaling Pathway * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• beta Catenin MeSH
• DVL2 protein, human MeSH Browser
• Dishevelled Proteins * MeSH

Polyglutamylation is a reversible posttranslational modification that is catalyzed by enzymes of the tubulin tyrosine ligase-like (TTLL) family. Here, we found that TTLL11 generates a previously unknown type of polyglutamylation that is initiated by the addition of a glutamate residue to the free C-terminal carboxyl group of a substrate protein. TTLL11 efficiently polyglutamylates the Wnt signaling protein Dishevelled 3 (DVL3), thereby changing the interactome of DVL3. Polyglutamylation increases the capacity of DVL3 to get phosphorylated, to undergo phase separation, and to act in the noncanonical Wnt pathway. Both carboxy-terminal polyglutamylation and the resulting reduction in phase separation capacity of DVL3 can be reverted by the deglutamylating enzyme CCP6, demonstrating a causal relationship between TTLL11-mediated polyglutamylation and phase separation. Thus, C-terminal polyglutamylation represents a new type of posttranslational modification, broadening the range of proteins that can be modified by polyglutamylation and providing the first evidence that polyglutamylation can modulate protein phase separation.

• Keywords
• Dishevelled 3, Noncanonical Wnt Signaling, Polyglutamylation, Protein Condensates, TTLL11,
• MeSH
• Phosphorylation MeSH
• HEK293 Cells MeSH
• Polyglutamic Acid metabolism   analogs & derivatives   MeSH
• Humans MeSH
• Peptide Synthases * metabolism   genetics   MeSH
• Protein Processing, Post-Translational * MeSH
• Dishevelled Proteins * metabolism   genetics   MeSH
• Phase Separation MeSH
• Wnt Signaling Pathway MeSH
• Signal Transduction MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• DVL3 protein, human MeSH Browser
• Polyglutamic Acid MeSH
• Peptide Synthases * MeSH
• Dishevelled Proteins * MeSH
• tubulin polyglutamylase MeSH Browser

This study introduces a novel cost-effective technique for cloning of linear DNA plasmid inserts, aiming to address the associated expenses linked with popular in vitro DNA assembly methods. Specifically, we introduce ECOLI (Efficient Cloning Of Linear Inserts), a method utilizing a PCR product-based site-directed mutagenesis. In comparison to other established in vitro DNA assembly methods, our approach is without the need for costly synthesis or specialized kits for recombination or restriction sites. ECOLI offers a fast, efficient, and economical alternative for cloning inserts up to several hundred nucleotides into plasmid constructs, thus enhancing cloning accessibility and efficiency. This method can enhance molecular biology research, as we briefly demonstrated on the Dishevelled gene from the WNT signaling pathway.

• Keywords
• Dishevelled, DNA cloning, In vitro DNA assembly, Mutagenesis, PCR, Plasmid-based cloning, Site-directed mutagenesis,
• MeSH
• DNA genetics   MeSH
• Cloning, Molecular * methods   MeSH
• Mutagenesis, Site-Directed * methods   MeSH
• Plasmids * genetics   MeSH
• Polymerase Chain Reaction methods   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• DNA MeSH