Cingulin binds to the ZU5 domain of scaffolding protein ZO-1 to promote its extended conformation, stabilization, and tight junction accumulation

• Published in: The Journal of biological chemistry Vol. 298; no. 4; p. 101797
• Main Authors: Vasileva, Ekaterina, Spadaro, Domenica, Rouaud, Florian, King, Jonathan M., Flinois, Arielle, Shah, Jimit, Sluysmans, Sophie, Méan, Isabelle, Jond, Lionel, Turner, Jerrold R., Citi, Sandra
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.04.2022; American Society for Biochemistry and Molecular Biology
• Subjects: cingulin; Cytoskeletal Proteins - chemistry; Cytoskeletal Proteins - genetics; Cytoskeletal Proteins - metabolism; Cytoskeleton - metabolism; Gene Knockdown Techniques; paracingulin; Phosphoproteins - genetics; Phosphoproteins - metabolism; Protein Domains; Protein Folding; Protein Stability; Protein Structure, Quaternary; tight junctions; Tight Junctions - metabolism; ZO-1; Zonula Occludens-1 Protein - chemistry; Zonula Occludens-1 Protein - genetics; Zonula Occludens-1 Protein - metabolism; cingulin; RT; FL; BSA; DMEM; FBS; IB; FRAP; tight junctions; MDCK; IF; CGNL1; eGFP; mCCD; paracingulin; ZA; AJ; GEF; GST; CGN; PLEKHA7; HEK; ZO-1; ZO; SIM; TJ; HA; ZIM
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/j.jbc.2022.101797

Zonula occludens-1 (ZO-1), the major scaffolding protein of tight junctions (TJs), recruits the cytoskeleton-associated proteins cingulin (CGN) and paracingulin (CGNL1) to TJs by binding to their N-terminal ZO-1 interaction motif. The conformation of ZO-1 can be either folded or extended, depending on cytoskeletal tension and intramolecular and intermolecular interactions, and only ZO-1 in the extended conformation recruits the transcription factor DbpA to TJs. However, the sequences of ZO-1 that interact with CGN and CGNL1 and the role of TJ proteins in ZO-1 TJ assembly are not known. Here, we used glutathione-S-transferase pulldowns and immunofluorescence microscopy to show that CGN and CGNL1 bind to the C-terminal ZU5 domain of ZO-1 and that this domain is required for CGN and CGNL1 recruitment to TJs and to phase-separated ZO-1 condensates in cells. We show that KO of CGN, but not CGNL1, results in decreased accumulation of ZO-1 at TJs. Furthermore, ZO-1 lacking the ZU5 domain showed decreased accumulation at TJs, was detectable along lateral contacts, had a higher mobile fraction than full-length ZO-1 by fluorescence recovery after photobleaching analysis, and had a folded conformation, as determined by structured illumination microscopy of its N-terminal and C-terminal ends. The CGN–ZU5 interaction promotes the extended conformation of ZO-1, since binding of the CGN–ZO-1 interaction motif region to ZO-1 resulted in its interaction with DbpA in cells and in vitro. Together, these results show that binding of CGN to the ZU5 domain of ZO-1 promotes ZO-1 stabilization and accumulation at TJs by promoting its extended conformation.