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1.
J Cell Sci ; 137(5)2024 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-37013686

RESUMO

Paracingulin (CGNL1) is recruited to tight junctions (TJs) by ZO-1 and to adherens junctions (AJs) by PLEKHA7. PLEKHA7 has been reported to bind to the microtubule minus-end-binding protein CAMSAP3, to tether microtubules to the AJs. Here, we show that knockout (KO) of CGNL1, but not of PLEKHA7, results in the loss of junctional CAMSAP3 and its redistribution into a cytoplasmic pool both in cultured epithelial cells in vitro and mouse intestinal epithelium in vivo. In agreement, GST pulldown analyses show that CGNL1, but not PLEKHA7, interacts strongly with CAMSAP3, and the interaction is mediated by their respective coiled-coil regions. Ultrastructure expansion microscopy shows that CAMSAP3-capped microtubules are tethered to junctions by the ZO-1-associated pool of CGNL1. The KO of CGNL1 results in disorganized cytoplasmic microtubules and irregular nuclei alignment in mouse intestinal epithelial cells, altered cyst morphogenesis in cultured kidney epithelial cells, and disrupted planar apical microtubules in mammary epithelial cells. Together, these results uncover new functions of CGNL1 in recruiting CAMSAP3 to junctions and regulating microtubule cytoskeleton organization and epithelial cell architecture.


Assuntos
Microtúbulos , Junções Íntimas , Animais , Camundongos , Junções Aderentes/metabolismo , Citoplasma/metabolismo , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Células Epiteliais/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Junções Íntimas/metabolismo
2.
J Biol Chem ; 298(4): 101797, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35259394

RESUMO

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.


Assuntos
Proteínas do Citoesqueleto , Junções Íntimas , Proteína da Zônula de Oclusão-1 , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Técnicas de Silenciamento de Genes , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Domínios Proteicos , Dobramento de Proteína , Estabilidade Proteica , Estrutura Quaternária de Proteína , Junções Íntimas/metabolismo , Proteína da Zônula de Oclusão-1/química , Proteína da Zônula de Oclusão-1/genética , Proteína da Zônula de Oclusão-1/metabolismo
3.
J Cell Biol ; 222(7)2023 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-37204781

RESUMO

The mechanisms that regulate the spatial sorting of nonmuscle myosins-2 (NM2) isoforms and couple them mechanically to the plasma membrane are unclear. Here we show that the cytoplasmic junctional proteins cingulin (CGN) and paracingulin (CGNL1) interact directly with NM2s through their C-terminal coiled-coil sequences. CGN binds strongly to NM2B, and CGNL1 to NM2A and NM2B. Knockout (KO), exogenous expression, and rescue experiments with WT and mutant proteins show that the NM2-binding region of CGN is required for the junctional accumulation of NM2B, ZO-1, ZO-3, and phalloidin-labeled actin filaments, and for the maintenance of tight junction membrane tortuosity and apical membrane stiffness. CGNL1 expression promotes the junctional accumulation of both NM2A and NM2B and its KO results in myosin-dependent fragmentation of adherens junction complexes. These results reveal a mechanism for the junctional localization of NM2A and NM2B and indicate that, by binding to NM2s, CGN and CGNL1 mechanically couple the actomyosin cytoskeleton to junctional protein complexes to mechanoregulate the plasma membrane.


Assuntos
Membrana Celular , Proteínas do Citoesqueleto , Citoesqueleto , Miosinas , Junções Aderentes/metabolismo , Membrana Celular/metabolismo , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Miosinas/metabolismo , Junções Íntimas/metabolismo
4.
Biochim Biophys Acta Biomembr ; 1862(10): 183399, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32553946

RESUMO

Tight and adherens junctions are specialized sites of cell-cell interaction in epithelia and endothelia, and are involved in barrier, adhesion, and signaling functions. These functions are orchestrated by a highly organized meshwork of macromolecules in the membrane and cytoplasmic compartments. In this review, we discuss the structural organization and functions of the major cytoplasmic scaffolding and adaptor proteins of vertebrate apical junctions (ZO proteins, afadin, PLEKHA7, cingulin, paracingulin, polarity complex proteins, and a few others), focusing on their interactions with cytoskeletal and signaling proteins. Furthermore, we discuss recent results highlighting how mechanical tension, protein-protein interactions and post-translational modifications regulate the conformation and function of scaffolding proteins, and how spontaneous phase separation into biomolecular condensates contributes to apical junction assembly. Using a sequence-based algorithm, a large fraction of cytoplasmic proteins of apical junctions are predicted to be phase separating proteins (PSPs), suggesting that formation of biomolecular condensates is a general mechanism to organize cell-cell contacts by clustering proteins.


Assuntos
Junções Intercelulares/metabolismo , Vertebrados/metabolismo , Animais , Fenômenos Biofísicos , Citoplasma/metabolismo , Ligantes
5.
Nat Commun ; 10(1): 1969, 2019 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-31036808

RESUMO

Long noncoding RNAs (lncRNAs) are emerging as regulators of fundamental biological processes. Here we report on the characterization of an intergenic lncRNA expressed in epithelial tissues which we termed EPR (Epithelial cell Program Regulator). EPR is rapidly downregulated by TGF-ß and its sustained expression largely reshapes the transcriptome, favors the acquisition of epithelial traits, and reduces cell proliferation in cultured mammary gland cells as well as in an animal model of orthotopic transplantation. EPR generates a small peptide that localizes at epithelial cell junctions but the RNA molecule per se accounts for the vast majority of EPR-induced gene expression changes. Mechanistically, EPR interacts with chromatin and regulates Cdkn1a gene expression by affecting both its transcription and mRNA decay through its association with SMAD3 and the mRNA decay-promoting factor KHSRP, respectively. We propose that EPR enables epithelial cells to control proliferation by modulating waves of gene expression in response to TGF-ß.


Assuntos
Estabilidade de RNA/genética , RNA Longo não Codificante/genética , Proteína Smad3/metabolismo , Transcriptoma/genética , Fator de Crescimento Transformador beta/farmacologia , Animais , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Humanos , RNA Longo não Codificante/efeitos dos fármacos
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