RESUMO
Genome editing is a powerful tool for establishing gene knockout or mutant cell lines. Here, we present a protocol for establishing knockout cell clones by deletion of large gene fragments using CRISPR-Cas9 with multiple guide RNAs. We describe steps for designing guide RNAs, cloning them into CRISPR-Cas9 vectors, cell seeding, transfection into cultured cells, clonal selection, and screening assays. This protocol can delete gene regions over 100 kbp, including GC-rich domains, and is applicable to various cell lines. For complete details on the use and execution of this protocol, please refer to Saito et al.,1 Saito and Endo et al.,2 and Higashi et al.3.
RESUMO
Epithelial tissues cover the surfaces and lumens of the internal organs of multicellular animals and crucially contribute to internal environment homeostasis by delineating distinct compartments within the body. This vital role is known as epithelial barrier function. Epithelial cells are arranged like cobblestones and intricately bind together to form an epithelial sheet that upholds this barrier function. Central to the restriction of solute and fluid diffusion through intercellular spaces are occluding junctions, tight junctions in vertebrates and septate junctions in invertebrates. As part of epithelial tissues, cells undergo constant renewal, with older cells being replaced by new ones. Simultaneously, the epithelial tissue undergoes relative rearrangement, elongating, and shifting directionally as a whole. The movement or shape changes within the epithelial sheet necessitate significant deformation and reconnection of occluding junctions. Recent advancements have shed light on the intricate mechanisms through which epithelial cells sustain their barrier function in dynamic environments. This review aims to introduce these noteworthy findings and discuss some of the questions that remain unanswered.
Assuntos
Células Epiteliais , Junções Íntimas , Animais , Humanos , Células Epiteliais/metabolismo , Células Epiteliais/citologia , Junções Íntimas/metabolismo , Junções Íntimas/fisiologia , Epitélio/metabolismo , Epitélio/fisiologiaRESUMO
Podocytes serve as part of the renal filtration unit with slit diaphragms. Although the structure of slit diaphragms between two cells is well characterized, how the tricellular contact of podocytes is organized and how it changes in injured podocytes remains unknown. This study focused on a tricellular junction protein, angulin-3, and its localization in healthy podocytes, in developmental stages, and in pathologic conditions, using a newly established monoclonal antibody. Angulin-3 was confined at tricellular junctions of primordial podocytes, then transiently localized at bicellular junctions as foot process interdigitation developed and the intercellular junctions rearranged into slit diaphragm, and eventually distributed in a sparse punctate pattern on the foot processes of adult podocytes. In the rodent podocyte injury models, angulin-3 showed bicellular localization between the foot processes, and the localization turned from punctate to dashed linear pattern along the effaced foot processes with the progression of podocyte injury. Angulin-3 also accumulated between foot processes in a linear pattern in kidney biopsy samples of human nephrotic syndrome. Additionally, the line length of angulin-3 staining signal correlated with risk of relapse under glucocorticoid therapy in patients with minimal change nephrotic syndrome. This study proposes an image program to score the linearity of the accumulation pattern of angulin-3 to evaluate the relapse risk of patients with minimal change nephrotic syndrome.
Assuntos
Nefrose Lipoide , Podócitos , Adulto , Humanos , Podócitos/metabolismo , Junções Íntimas/patologia , Nefrose Lipoide/metabolismo , Nefrose Lipoide/patologia , Junções Intercelulares/metabolismo , RecidivaRESUMO
Epithelial barrier function is commonly analyzed using transepithelial electrical resistance, which measures ion flux across a monolayer, or by adding traceable macromolecules and monitoring their passage across the monolayer. Although these methods measure changes in global barrier function, they lack the sensitivity needed to detect local or transient barrier breaches, and they do not reveal the location of barrier leaks. Therefore, we previously developed a method that we named the zinc-based ultrasensitive microscopic barrier assay (ZnUMBA), which overcomes these limitations, allowing for detection of local tight junction leaks with high spatiotemporal resolution. Here, we present expanded applications for ZnUMBA. ZnUMBA can be used in Xenopus embryos to measure the dynamics of barrier restoration and actin accumulation following laser injury. ZnUMBA can also be effectively utilized in developing zebrafish embryos as well as cultured monolayers of Madin-Darby canine kidney (MDCK) II epithelial cells. ZnUMBA is a powerful and flexible method that, with minimal optimization, can be applied to multiple systems to measure dynamic changes in barrier function with spatiotemporal precision.
Assuntos
Células Epiteliais , Zinco , Animais , Cães , Peixe-Zebra , Células Madin Darby de Rim Canino , Junções Íntimas , ActinasRESUMO
Tight junctions (TJs) are cell-cell junction structures critical for controlling paracellular permeability. On freeze-fracture replica electron microscopy, they appear as a continuous network of fibrils (TJ strands). TJ strands function as zippers that create a physical barrier against paracellular diffusion of molecules. The morphology of the TJ strand network varies greatly between tissues, and in recent years, studies have highlighted the mechanisms regulating the morphology of TJ strand networks and on their relevance to barrier function. In this review, we discuss evidence regarding the components of the TJ strand and the mechanisms for creating the TJ strand network. Furthermore, we discuss and hypothesize how its morphology contributes to the establishment of the epithelial barrier.
Assuntos
Epitélio , Junções Íntimas , Junções Íntimas/química , Junções Íntimas/fisiologiaRESUMO
TJs maintain the epithelial barrier by regulating paracellular permeability. Since TJs are under dynamically fluctuating intercellular tension, cells must continuously survey and repair any damage. However, the underlying mechanisms allowing cells to sense TJ damage and repair the barrier are not yet fully understood. Here, we showed that proteinases play an important role in the maintenance of the epithelial barrier. At TJ break sites, EpCAM-claudin-7 complexes on the basolateral membrane become accessible to apical membrane-anchored serine proteinases (MASPs) and the MASPs cleave EpCAM. Biochemical data and imaging analysis suggest that claudin-7 released from EpCAM contributes to the rapid repair of damaged TJs. Knockout (KO) of MASPs drastically reduced barrier function and live-imaging of TJ permeability showed that MASPs-KO cells exhibited increased size, duration, and frequency of leaks. Together, our results reveal a novel mechanism of TJ maintenance through the localized proteolysis of EpCAM at TJ leaks, and provide a better understanding of the dynamic regulation of epithelial permeability.
Assuntos
Claudinas , Molécula de Adesão da Célula Epitelial , Serina Proteases Associadas a Proteína de Ligação a Manose , Junções Íntimas , Claudinas/genética , Claudinas/metabolismo , Molécula de Adesão da Célula Epitelial/genética , Molécula de Adesão da Célula Epitelial/metabolismo , Serina Proteases Associadas a Proteína de Ligação a Manose/metabolismo , Proteólise , Junções Íntimas/metabolismo , Técnicas de Inativação de GenesRESUMO
The tightjunction protein claudin9 (CLDN9) is barely distributed in normal adult tissues but is ectopically expressed in various cancer types. Although multiple databases indicated upregulation of CLDN9 in endometrial cancers at the mRNA level, its protein expression and biological roles remain obscure. In the present study, the prognostic significance of CLDN9 expression in endometrial cancer was evaluated by immunohistochemical staining and semiquantification using formalinfixed paraffinembedded specimens obtained from 248 endometrial carcinoma cases. A total of 43 cases (17.3%) had high CLDN9 expression, whereas 205 cases (82.7%) exhibited low CLDN9 expression. The 5year diseasespecific survival rates in the high and low CLDN9 expression groups were 62.8 and 87.8% (P<0.001), respectively. In addition, multivariate analysis revealed that high CLDN9 expression was an independent prognostic factor (hazard ratio, 4.99; 95% CI, 1.9612.70; P<0.001). Furthermore, CLDN9 expression was significantly correlated with the expression of CLDN6 (P<0.001), which is the closest CLDN member to CLDN9 and a poor prognostic factor for endometrial carcinoma. The 5year diseasespecific survival rate of cases with CLDN6high/CLDN9high, CLDN6high/CLDN9low and CLDN6low/CLDN9high status was 30.0, 37.5 and 72.7%, respectively, whereas that of CLDN6low/CLDN9low was 89.8% (P=0.004). In conclusion, aberrant CLDN9 expression is a predictor of poor prognosis for endometrial cancer and may be utilized in combination with CLDN6 to achieve higher sensitivity.
Assuntos
Claudinas , Neoplasias do Endométrio , Biomarcadores , Claudinas/genética , Claudinas/metabolismo , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/patologia , Feminino , Formaldeído , Humanos , Prognóstico , RNA Mensageiro/metabolismoRESUMO
Occludin, tricellulin, and marvelD3 belong to the tight junction (TJ)-associated MARVEL protein family. Occludin and tricellulin jointly contribute to TJ strand branching point formation and epithelial barrier maintenance. However, whether marvelD3 has the same function remains unclear. Furthermore, the roles of the carboxy-terminal cytoplasmic tail, which is conserved in occludin and tricellulin, on the regulation of TJ strand morphology have not yet been explored in epithelial cells. We established tricellulin/occludin/marveld3 triple-gene knockout (tKO) MDCK II cells and evaluated the roles of marvelD3 in the TJ strand structure and barrier function using MDCK II cells and a mathematical model. The complexity of TJ strand networks and paracellular barrier did not change in tKO cells compared to that in tricellulin/occludin double-gene knockout (dKO) cells. Exogenous marvelD3 expression in dKO cells did not increase the complexity of TJ strand networks and epithelial barrier tightness. The expression of the carboxy-terminal truncation mutant of tricellulin restored the barrier function in the dKO cells, whereas occludin lacking the carboxy-terminal cytoplasmic tail was not expressed on the plasma membrane. These data suggest that marvelD3 does not affect the morphology of TJ strands and barrier function in MDCK II cells and that the carboxy-terminal cytoplasmic tail of tricellulin is dispensable for barrier improvement.
Assuntos
Proteína 2 com Domínio MARVEL , Junções Íntimas , Humanos , Cães , Animais , Junções Íntimas/metabolismo , Ocludina/genética , Ocludina/metabolismo , Proteína 2 com Domínio MARVEL/metabolismo , Células Epiteliais/metabolismo , Proteínas de Junções Íntimas/metabolismo , Células Madin Darby de Rim CaninoRESUMO
The anterior pituitary gland regulates growth, metabolism, and reproduction by secreting hormones. Folliculo-stellate (FS) cells are non-endocrine cells located among hormone-producing cells in the anterior pituitary glands. They form follicular lumens, which are sealed by tight junctions (TJs). Although FS cells are hypothesized to contribute to fine-tuning of endocrine cells, little is known about the exact roles of FS cells. Here, we investigated the molecular composition of TJs in FS cells. We demonstrated that occludin is a good marker for TJs in the pituitary gland and examined the structure of the lumens surrounded by FS cells. We also found that claudin-9 is a major component of TJs in the FS cells. In immunoelectron microscopy, claudin-9 was specifically localized at TJs of the FS cells. The expression of claudin-9 was gradually increased in the pituitary gland after birth, suggesting that claudin-9 is developmentally regulated and performs some specific functions on the paracellular barrier of follicles in the pituitary gland. Furthermore, we found that angulin-1, angulin-2, and tricellulin are localized at the tricellular contacts of the FS cells. Our findings provide a first comprehensive molecular profile of TJs in the FS cells, and may lead us towards unveiling the FS cell functions.
Assuntos
Claudinas/metabolismo , Adeno-Hipófise/citologia , Adeno-Hipófise/metabolismo , Animais , Astrócitos/metabolismo , Fenômenos Fisiológicos Celulares , Claudinas/fisiologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ocludina/metabolismo , Hipófise/metabolismo , Adeno-Hipófise/fisiologia , Junções Íntimas/metabolismo , Junções Íntimas/fisiologiaRESUMO
Malignant mesothelioma is a cancer with a poor survival rate. It is difficult to diagnose mesotheliomas because they show a variety of histological patterns similar to those of various other cancers. However, since currently used positive markers for mesotheliomas may show false positives or false negatives, a novel mesothelial positive marker is required. In the present study, we screened 25 claudins and found that claudin-15 is expressed in the mesothelial cells. We made new rat anti-human claudin-15 (CLDN15) monoclonal antibodies that selectively recognize CLDN15, and investigated whether CLDN15 is a good positive marker for malignant pleural mesotheliomas (MPMs) using MPM tissue samples by immunohistochemistry and semi-quantification of the expression level using an immunoreactive score (IRS) method. Of 42 MPM samples, 83% were positive for CLDN15. The positive ratio was equal to or greater than other positive markers for MPMs including calretinin (81%), WT-1 (50%), and D2-40 (81%). In 50 lung adenocarcinoma sections, four cases were positive for CLDN15 and the specificity (92%) was comparable with other markers (90-100%). Notably, CLDN15 was rarely detected in 24 non-mesothelial tumors in the tissue microarray (12/327 cases). In conclusion, CLDN15 can be used in the clinical setting as a positive marker for MPM diagnosis.
Assuntos
Adenocarcinoma de Pulmão/diagnóstico , Calbindina 2/genética , Claudinas/genética , Mesotelioma Maligno/diagnóstico , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Biomarcadores Tumorais/genética , Diagnóstico Diferencial , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Mesotelioma Maligno/genética , Mesotelioma Maligno/patologia , Pessoa de Meia-Idade , Ratos , Proteínas WT1/genéticaRESUMO
Tight junctions (TJs) are composed of a claudin-based anastomosing network of TJ strands at which plasma membranes of adjacent epithelial cells are closely attached to regulate the paracellular permeability. Although the TJ proteins occludin and tricellulin have been known to be incorporated in the TJ strand network, their molecular functions remain unknown. Here, we established tricellulin/occludin-double knockout (dKO) MDCK II cells using a genome editing technique and evaluated the structure and barrier function of these cells. In freeze-fracture replica electron microscopy, the TJ strands of tricellulin/occludin-dKO cells had fewer branches and were less anastomosed compared with the controls. The paracellular permeability of ions and small tracers was increased in the dKO cells. A single KO of tricellulin or occludin had limited effects on the morphology and permeability of TJs. Mathematical simulation using a simplified TJ strand network model predicted that reduced cross-links in TJ strands lead to increased permeability of ions and small macromolecules. Furthermore, overexpression of occludin increased the complexity of TJ strand network and strengthened barrier function. Taken together, our data suggest that tricellulin and occludin mediate the formation and/or stabilization of TJ-strand branching points and contribute to the maintenance of epithelial barrier integrity.
Assuntos
Proteína 2 com Domínio MARVEL/metabolismo , Ocludina/metabolismo , Junções Íntimas/metabolismo , Animais , Linhagem Celular , Claudinas/metabolismo , Cães , Células Epiteliais/metabolismo , Células HEK293 , Humanos , Proteína 2 com Domínio MARVEL/fisiologia , Células Madin Darby de Rim Canino , Ocludina/fisiologia , Junções Íntimas/fisiologiaRESUMO
Tricellular tight junctions (tTJs) create paracellular barriers at tricellular contacts (TCs), where the vertices of three polygonal epithelial cells meet. tTJs are marked by the enrichment of two types of membrane proteins, tricellulin and angulin family proteins. However, how TC geometry is recognized for tTJ formation remains unknown. In the present study, we examined the molecular mechanism for the assembly of angulin-1 at the TCs. We found that clusters of cysteine residues in the juxtamembrane region within the cytoplasmic domain of angulin-1 are highly palmitoylated. Mutagenesis analyses of the cysteine residues in this region revealed that palmitoylation is essential for localization of angulin-1 at TCs. Consistently, suppression of Asp-His-His-Cys motif-containing palmitoyltransferases expressed in EpH4 cells significantly impaired the TC localization of angulin-1. Cholesterol depletion from the plasma membrane of cultured epithelial cells hampered the localization of angulin-1 at TCs, suggesting the existence of a lipid membrane microdomain at TCs that attracts highly palmitoylated angulin-1. Furthermore, the extracellular domain of angulin-1 was also required for its TC localization, irrespective of the intracellular palmitoylation. Taken together, our findings suggest that both angulin-1's extracellular domain and palmitoylation of its cytoplasmic region are required for its assembly at TCs.
Assuntos
Colesterol/genética , Lipoilação/genética , Microdomínios da Membrana/genética , Receptores de Lipoproteínas/genética , Comunicação Celular/genética , Colesterol/metabolismo , Cisteína/química , Cisteína/genética , Células Epiteliais/metabolismo , Humanos , Junções Intercelulares/genética , Proteína 2 com Domínio MARVEL , Microdomínios da Membrana/química , Domínios Proteicos/genética , Processamento de Proteína Pós-Traducional/genética , Receptores de Lipoproteínas/química , Junções Íntimas/genética , Junções Íntimas/metabolismoRESUMO
Tricellular junctions are specialized cell-cell junctions formed at sites where three epithelial or endothelial cells make contact at their apical side. By holding three cells together, tricellular junctions contribute to the maintenance of epithelial barrier function and mechanical integrity. In addition, recent studies have uncovered new functions of tricellular junctions at both cellular and physiological levels. In this review, we describe the architecture and molecular components of tricellular junctions and discuss how tricellular junctions participate in various biological processes.
Assuntos
Junções Aderentes/metabolismo , Desmossomos/metabolismo , Junções Íntimas/metabolismo , Junções Aderentes/química , Animais , Desmossomos/química , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Células Epiteliais/fisiologia , Humanos , Proteínas de Junções Íntimas/metabolismo , Junções Íntimas/químicaRESUMO
Cell adhesion is essential for proper tissue architecture and function in multicellular organisms. Cell adhesion molecules not only maintain tissue integrity but also possess signaling properties that contribute to diverse cellular events such as cell growth, survival, differentiation, polarity, and migration; however, the underlying molecular basis remains poorly defined. Here we identify that the cell adhesion signal initiated by the tight-junction protein claudin-6 (CLDN6) regulates nuclear receptor activity. We show that CLDN6 recruits and activates Src-family kinases (SFKs) in second extracellular domain-dependent and Y196/200-dependent manners, and SFKs in turn phosphorylate CLDN6 at Y196/200. We demonstrate that the CLDN6/SFK/PI3K/AKT axis targets the AKT phosphorylation sites in the retinoic acid receptor γ (RARγ) and the estrogen receptor α (ERα) and stimulates their activities. Interestingly, these phosphorylation motifs are conserved in 14 of 48 members of human nuclear receptors. We propose that a similar link between diverse cell adhesion and nuclear receptor signalings coordinates a wide variety of physiological and pathological processes.
Assuntos
Adesão Celular/fisiologia , Claudinas/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Linhagem Celular , Claudinas/genética , Receptor alfa de Estrogênio/metabolismo , Regulação da Expressão Gênica , Humanos , Camundongos , Mutação , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Domínios Proteicos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores do Ácido Retinoico/genética , Receptores do Ácido Retinoico/metabolismo , Transdução de Sinais , Tirosina/genética , Quinases da Família src/metabolismo , Receptor gama de Ácido RetinoicoRESUMO
Tight junctions contribute to epithelial barrier function by selectively regulating the quantity and type of molecules that cross the paracellular barrier. Experimental approaches to evaluate the effectiveness of tight junctions are typically global, tissue-scale measures. Here, we introduce Zinc-based Ultrasensitive Microscopic Barrier Assay (ZnUMBA), which we used in Xenopus laevis embryos to visualize short-lived, local breaches in epithelial barrier function. These breaches, or leaks, occur as cell boundaries elongate, correspond to visible breaks in the tight junction, and are followed by transient localized Rho activation, or Rho flares. We discovered that Rho flares restore barrier function by driving concentration of tight junction proteins through actin polymerization and ROCK-mediated localized contraction of the cell boundary. We conclude that Rho flares constitute a damage control mechanism that reinstates barrier function when tight junctions become locally compromised because of normally occurring changes in cell shape and tissue tension.
Assuntos
Junções Aderentes/metabolismo , Células Epiteliais/metabolismo , Proteínas de Membrana/metabolismo , Junções Íntimas/metabolismo , Quinases Associadas a rho/metabolismo , Actinas/metabolismo , Animais , Células CACO-2/citologia , Humanos , Fosfoproteínas/metabolismo , Junções Íntimas/patologia , Xenopus laevis/metabolismoRESUMO
Cellular forces sculpt organisms during development, while misregulation of cellular mechanics can promote disease. Here, we investigate how the actomyosin scaffold protein anillin contributes to epithelial mechanics in Xenopus laevis embryos. Increased mechanosensitive recruitment of vinculin to cell-cell junctions when anillin is overexpressed suggested that anillin promotes junctional tension. However, junctional laser ablation unexpectedly showed that junctions recoil faster when anillin is depleted and slower when anillin is overexpressed. Unifying these findings, we demonstrate that anillin regulates medial-apical actomyosin. Medial-apical laser ablation supports the conclusion that that tensile forces are stored across the apical surface of epithelial cells, and anillin promotes the tensile forces stored in this network. Finally, we show that anillin's effects on cellular mechanics impact tissue-wide mechanics. These results reveal anillin as a key regulator of epithelial mechanics and lay the groundwork for future studies on how anillin may contribute to mechanical events in development and disease.
Assuntos
Actomiosina/metabolismo , Proteínas Contráteis/metabolismo , Células Epiteliais/metabolismo , Actinas , Trifosfato de Adenosina/farmacologia , Animais , Fenômenos Biomecânicos , Polaridade Celular , Proteínas Contráteis/química , Embrião não Mamífero/metabolismo , Miosina Tipo II/metabolismo , Ligação Proteica , Domínios Proteicos , Estabilidade Proteica , Vinculina/metabolismo , Xenopus laevis/embriologia , Xenopus laevis/metabolismoRESUMO
Reorganization of the actin cytoskeleton is crucial for cellular processes, including cytokinesis and cell-cell junction remodeling. Formins are conserved processive actin-polymerizing machines that regulate actin dynamics by nucleating, elongating, and bundling linear actin filaments. Because the formin family is large, with at least 15 members in vertebrates, there have not been any comprehensive studies examining formin localization and function within a common cell type. Here, we characterized the localization of all 15 formins in epithelial cells of Xenopus laevis gastrula-stage embryos. Dia1 and Dia2 localized to tight junctions, while Fhod1 and Fhod3 localized to adherens junctions. Only Dia3 strongly localized at the cytokinetic contractile ring. The Diaphanous inhibitory domain-dimerization domain (DID-DD) region of Dia1 was sufficient for Dia1 localization, and overexpression of a Dia1 DID-DD fragment competitively removed Dia1 and Dia2 from cell-cell junctions. In Dia1 DID-DD-overexpressing cells, Dia1 and Dia2 were mislocalized to the contractile ring, and cells exhibited increased cytokinesis failure. This work provides a comprehensive analysis of the localization of all 15 vertebrate formins in epithelial cells and suggests that misregulated formin localization results in epithelial cytokinesis failure.
Assuntos
Células Epiteliais/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo , Junções Aderentes/metabolismo , Animais , Sequência Conservada , Citocinese , Proteínas de Fluorescência Verde/metabolismo , Transporte Proteico , Junções Íntimas/metabolismoRESUMO
Tricellular contacts are the places where three cells meet. In vertebrate epithelial cells, specialized structures called tricellular tight junctions (tTJs) and tricellular adherens junctions (tAJs) have been identified. tTJs are important for the maintenance of barrier function, and disruption of tTJ proteins contributes to familial deafness. tAJs have recently been attracting the attention of mechanobiologists because these sites are hot spots of epithelial tension. Although the molecular components, regulation, and function of tTJs and tAJs, as well as of invertebrate tricellular junctions, are beginning to be characterized, many questions remain. Here we broadly cover what is known about tricellular junctions, propose a new model for tension transmission at tAJs, and discuss key open questions.
Assuntos
Junções Íntimas/metabolismo , Junções Aderentes/metabolismo , Animais , Surdez/metabolismo , Células Epiteliais/metabolismo , Humanos , Proteína 2 com Domínio MARVEL/metabolismo , Proteínas de Membrana/metabolismo , Transporte Proteico/fisiologiaRESUMO
Epithelial integrity and barrier function must be maintained during the complex cell shape changes that occur during cytokinesis in vertebrate epithelial tissue. Here, we investigate how adherens junctions and bicellular and tricellular tight junctions are maintained and remodeled during cell division in the Xenopus laevis embryo. We find that epithelial barrier function is not disrupted during cytokinesis and is mediated by sustained tight junctions. Using fluorescence recovery after photobleaching (FRAP), we demonstrate that adherens junction proteins are stabilized at the cleavage furrow by increased tension. We find that Vinculin is recruited to the adherens junction at the cleavage furrow, and that inhibiting recruitment of Vinculin by expressing a dominant-negative mutant increases the rate of furrow ingression. Furthermore, we show that cells neighboring the cleavage plane are pulled between the daughter cells, making a new interface between neighbors, and two new tricellular tight junctions flank the midbody following cytokinesis. Our data provide new insight into how epithelial integrity and barrier function are maintained throughout cytokinesis in vertebrate epithelial tissue.
Assuntos
Junções Aderentes/metabolismo , Junções Íntimas/metabolismo , Xenopus laevis/fisiologia , Animais , Citocinese , Embrião não Mamífero , Epitélio/embriologia , Epitélio/crescimento & desenvolvimento , Recuperação de Fluorescência Após Fotodegradação , Proteínas de Junções Íntimas/metabolismo , Vinculina/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriologia , Xenopus laevis/crescimento & desenvolvimentoRESUMO
Localized activation of Rho GTPases is essential for multiple cellular functions, including cytokinesis and formation and maintenance of cell-cell junctions. Although MgcRacGAP (Mgc) is required for spatially confined RhoA-GTP at the equatorial cortex of dividing cells, both the target specificity of Mgc's GAP activity and the involvement of phosphorylation of Mgc at Ser-386 are controversial. In addition, Mgc's function at cell-cell junctions remains unclear. Here, using gastrula-stage Xenopus laevis embryos as a model system, we examine Mgc's role in regulating localized RhoA-GTP and Rac1-GTP in the intact vertebrate epithelium. We show that Mgc's GAP activity spatially restricts accumulation of both RhoA-GTP and Rac1-GTP in epithelial cells--RhoA at the cleavage furrow and RhoA and Rac1 at cell-cell junctions. Phosphorylation at Ser-386 does not switch the specificity of Mgc's GAP activity and is not required for successful cytokinesis. Furthermore, Mgc regulates adherens junction but not tight junction structure, and the ability to regulate adherens junctions is dependent on GAP activity and signaling via the RhoA pathway. Together these results indicate that Mgc's GAP activity down-regulates the active populations of RhoA and Rac1 at localized regions of epithelial cells and is necessary for successful cytokinesis and cell-cell junction structure.