1. Cingulin binds to the ZU5 domain of scaffolding protein ZO-1 to promote its extended conformation, stabilization, and tight junction accumulation.
- Author
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Vasileva E, Spadaro D, Rouaud F, King JM, Flinois A, Shah J, Sluysmans S, Méan I, Jond L, Turner JR, and Citi S
- Subjects
- Cytoskeleton metabolism, Gene Knockdown Techniques, Phosphoproteins genetics, Phosphoproteins metabolism, Protein Domains, Protein Folding, Protein Stability, Protein Structure, Quaternary, Cytoskeletal Proteins chemistry, Cytoskeletal Proteins genetics, Cytoskeletal Proteins metabolism, Tight Junctions metabolism, Zonula Occludens-1 Protein chemistry, Zonula Occludens-1 Protein genetics, Zonula Occludens-1 Protein metabolism
- 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., Competing Interests: Conflict of interest J. R. T. is a founder and shareholder of Thelium Therapeutics and has served as a consultant for Entrinsic, Immunic, and Kallyope. S. C. has served as a consultant for Immunic. All other authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2022
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