{"doi":"10.1016/j.jbc.2022.101797","title":"Cingulin binds to the ZU5 domain of scaffolding protein ZO-1 to promote its extended conformation, stabilization, and tight junction accumulation","abstract":"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.","journal":"Journal of Biological Chemistry","year":2022,"id":243059,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":35,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9591,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2022-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":600333,"name":"Domenica Spadaro","orcid":null,"position":1,"is_corresponding":false},{"id":875468,"name":"Florian Rouaud","orcid":null,"position":2,"is_corresponding":false},{"id":874758,"name":"Jonathan M. King","orcid":"0000-0003-1791-0636","position":3,"is_corresponding":false},{"id":874759,"name":"Arielle Flinois","orcid":"0000-0002-7763-5632","position":4,"is_corresponding":false},{"id":874760,"name":"Jimit Shah","orcid":"0000-0002-7217-1293","position":5,"is_corresponding":false},{"id":683647,"name":"Sophie Sluysmans","orcid":"0000-0002-6094-5482","position":6,"is_corresponding":false},{"id":683648,"name":"Isabelle Méan","orcid":"0000-0002-3453-0507","position":7,"is_corresponding":false},{"id":600334,"name":"Lionel Jond","orcid":null,"position":8,"is_corresponding":false},{"id":250828,"name":"Jerrold R. Turner","orcid":"0000-0003-0627-9455","position":9,"is_corresponding":false},{"id":599800,"name":"Sandra Citi","orcid":"0000-0002-6537-4818","position":10,"is_corresponding":false},{"id":874757,"name":"Ekaterina Vasileva","orcid":"0000-0003-1620-2048","position":0,"is_corresponding":true}],"reference_count":57,"raw_metadata":null,"created_at":"2026-07-19T00:23:16.032700Z","pmid":"35259394","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}