RESUMEN
To generate and maintain epithelial cell polarity, specific sorting of proteins into vesicles destined for the apical and basolateral domain is required. Syntaxin 3 and 4 are apical and basolateral SNARE proteins important for the specificity of vesicle fusion at the apical and basolateral plasma membrane domains, respectively, but how these proteins are specifically targeted to these domains themselves is unclear. Munc18/SM proteins are potential regulators of this process. Like syntaxins, they are crucial for exocytosis and vesicle fusion. However, how munc18c and syntaxin 4 regulate the function of each other is unclear. Here, we investigated the requirement of syntaxin 4 in the delivery of basolateral membrane and secretory proteins, the basolateral targeting of syntaxin 4, and the role of munc18c in this targeting. Depletion of syntaxin 4 resulted in significant reduction of basolateral targeting, suggesting no compensation by other syntaxin forms. Mutational analysis identified amino acids Leu-25 and to a lesser extent Val-26 as essential for correct localization of syntaxin 4. Recently, it was shown that the N-terminal peptide of syntaxin 4 is involved in binding to munc18c. A mutation in this region that affects munc18c binding shows that munc18c binding is required for stabilization of syntaxin 4 at the plasma membrane but not for its correct targeting. We conclude that the N terminus serves two functions in membrane targeting. First, it harbors the sorting motif, which targets syntaxin 4 basolaterally in a munc18c-independent manner and second, it allows for munc18c binding, which stabilizes the protein in a munc18c-dependent manner.
Asunto(s)
Membrana Celular/metabolismo , Exocitosis/fisiología , Proteínas Munc18/metabolismo , Proteínas Qa-SNARE/metabolismo , Animales , Membrana Celular/genética , Perros , Proteínas Munc18/genética , Mutación Missense , Péptidos/genética , Péptidos/metabolismo , Unión Proteica/fisiología , Estabilidad Proteica , Transporte de Proteínas/fisiología , Proteínas Qa-SNARE/genética , Proteínas SNARE/genética , Proteínas SNARE/metabolismoRESUMEN
Podocalyxin/Gp135 was recently demonstrated to participate in the formation of a preapical complex to set up initial polarity in MDCK cells, a function presumably depending on the apical targeting of Gp135. We show that correct apical sorting of Gp135 depends on a bipartite signal composed of an extracellular O-glycosylation-rich region and the intracellular PDZ domain-binding motif. The function of this PDZ-binding motif could be substituted with a fusion construct of Gp135 with Ezrin-binding phosphoprotein 50 (EBP50). In accordance with this observation, EBP50 binds to newly synthesized Gp135 at the Golgi apparatus and facilitates oligomerization and sorting of Gp135 into a clustering complex. A defective connection between Gp135 and EBP50 or EBP50 knockdown results in a delayed exit from the detergent-resistant microdomain, failure of oligomerization, and basolateral missorting of Gp135. Furthermore, the basolaterally missorted EBP50-binding defective mutant of Gp135 was rapidly retrieved via a PKC-dependent mechanism. According to these findings, we propose a model by which a highly negative charged transmembrane protein could be packed into an apical sorting platform with the aid of its cytoplasmic partner EBP50.
Asunto(s)
Sialoglicoproteínas/metabolismo , Secuencias de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/genética , Polaridad Celular , Células Cultivadas , Proteínas del Citoesqueleto/metabolismo , Perros , Endocitosis , Glicosilación , Microdominios de Membrana/metabolismo , Modelos Biológicos , Mutación , Proteína Quinasa C/metabolismo , Señales de Clasificación de Proteína/genética , Estructura Terciaria de Proteína , ARN Interferente Pequeño/genética , Sialoglicoproteínas/química , Sialoglicoproteínas/genética , Transducción de SeñalRESUMEN
The plasma membrane of epithelial cells has two physically separated membrane domains. This membrane polarization is essential for the function of epithelial cells. It has been well established that different plasma membrane syntaxin forms are expressed in epithelial cells. In addition, these syntaxin forms can have a polarized localization, suggesting that they may play a direct role in the specificity of polarized membrane delivery. To determine the mechanism of the polarized syntaxin localization, we have made several chimeras of syntaxin 3 and 4. This allowed us to identify the protein sequences involved in this polarized localization. Using this technique, we showed that targeting information of syntaxin 3 and 4 is located in the first 30 amino acids.
Asunto(s)
Bioensayo/métodos , Polaridad Celular , Células Epiteliales/metabolismo , Proteínas Qa-SNARE/metabolismo , Adenoviridae/genética , Animales , Western Blotting , Línea Celular , Clusterina/metabolismo , Perros , Electroforesis en Gel de Poliacrilamida , Técnica del Anticuerpo Fluorescente , Vectores Genéticos , Microscopía Confocal , Transporte de Proteínas , Proteínas Qa-SNARE/química , Proteínas Qa-SNARE/genética , Proteínas Recombinantes de Fusión/metabolismo , Factores de Tiempo , Transducción GenéticaRESUMEN
In polarized epithelial cells syntaxin 3 is at the apical plasma membrane and is involved in delivery of proteins from the trans-Golgi network to the apical surface. The highly related syntaxin 4 is at the basolateral surface. The complementary distribution of these syntaxins suggests that they play a role in the specificity of membrane traffic to the two surfaces. We constructed a chimeric syntaxin where we removed the N-terminal 29 residues of syntaxin 3 and replaced it with the corresponding portion of syntaxin 4. When expressed in polarized epithelial cells, this chimera was exclusively localized to the basolateral surface. This indicates that the N-terminal domain of syntaxin 3 contains information for its polarized localization. In contrast to the apical localization of syntaxin 3, the basolateral localization of syntaxin 4 was not dependent on its N-terminal domain. Syntaxin 3 normally binds to Munc18b, but not to the related Munc18c. Overexpression of the chimera together with overexpression of Munc18b caused membrane and secretory proteins that are normally sent primarily to the apical surface to exhibit increased delivery to the basolateral surface. We suggest that syntaxins may play a role in determining the specificity of membrane targeting by permitting fusion with only certain target membranes.
Asunto(s)
Células Epiteliales/fisiología , Proteínas Qa-SNARE/fisiología , Animales , Línea Celular , Polaridad Celular , Perros , Células Epiteliales/citología , TransfecciónRESUMEN
Classic cadherins are important regulators of tissue morphogenesis. The predominant cadherin in epithelial cells, E-cadherin, has been extensively studied because of its critical role in normal epithelial development and carcinogenesis. Epithelial cells may also coexpress other cadherins, but their roles are less clear. The Madin Darby canine kidney (MDCK) cell line has been a popular mammalian model to investigate the role of E-cadherin in epithelial polarization and tubulogenesis. However, MDCK cells also express relatively high levels of cadherin-6, and it is unclear whether the functions of this cadherin are redundant to those of E-cadherin. We investigate the specific roles of both cadherins using a knockdown approach. Although we find that both cadherins are able to form adherens junctions at the basolateral surface, we show that they have specific and mutually exclusive roles in epithelial morphogenesis. Specifically, we find that cadherin-6 functions as an inhibitor of tubulogenesis, whereas E-cadherin is required for lumen formation. Ablation of cadherin-6 leads to the spontaneous formation of tubules, which depends on increased phosphoinositide 3-kinase (PI3K) activity. In contrast, loss of E-cadherin inhibits lumen formation by a mechanism independent of PI3K.
Asunto(s)
Cadherinas/metabolismo , Túbulos Renales/embriología , Riñón/embriología , Uniones Adherentes/metabolismo , Animales , Cadherinas/genética , Cadherinas/inmunología , Adhesión Celular , Agregación Celular , Diferenciación Celular , Línea Celular , Polaridad Celular , Perros , Células Epiteliales/citología , Células Epiteliales/metabolismo , Riñón/citología , Riñón/metabolismo , Túbulos Renales/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Interferencia de ARNRESUMEN
It is crucial for organ homeostasis that epithelia have effective mechanisms to restrict motility and cell proliferation in order to maintain tissue architecture. On the other hand, epithelial cells need to rapidly and transiently acquire a more mesenchymal phenotype, with high levels of cell motility and proliferation, in order to repair epithelia upon injury. Cross talk between cell-cell and cell-matrix signaling is crucial for regulating these transitions. The Pak1-betaPIX-GIT complex is an effector complex downstream of the small GTPase Rac1. We previously showed that translocation of this complex from cell-matrix to cell-cell adhesion sites was required for the establishment of contact inhibition of proliferation. In this study, we provide evidence that this translocation depends on cadherin function. Cadherins do not recruit the complex by direct interaction. Rather, we found that inhibition of the normal function of cadherin or Pak1 leads to defects in focal adhesion turnover and to increased signaling by phosphatidylinositol 3-kinase. We propose that cadherins are involved in regulation of contact inhibition by controlling the function of the Pak1-betaPIX-GIT complex at focal contacts.
Asunto(s)
Cadherinas/metabolismo , Proliferación Celular , Inhibición de Contacto/fisiología , Matriz Extracelular/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Transducción de Señal/fisiología , Quinasas p21 Activadas/metabolismo , Uniones Adherentes/metabolismo , Animales , Cadherinas/genética , Línea Celular , Cromonas/metabolismo , Perros , Factores de Intercambio de Guanina Nucleótido/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Morfolinas/metabolismo , Complejos Multiproteicos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho , Quinasas p21 Activadas/genética , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rac1/metabolismoRESUMEN
Wound healing in epithelia requires coordinated cell migration and proliferation regulated by signaling mechanisms that are poorly understood. Here we show that epithelial cells expressing constitutively active or kinase-dead mutants of the Rac/Cdc42 effector Pak1 fail to undergo growth arrest upon wound closure. Strikingly, this phenotype is only observed when the Pak1 kinase mutants are expressed in cells possessing a free lateral surface, i.e. one that is not engaged in contact with neighboring cells. The Pak1 kinase mutants perturb contact inhibition by a mechanism that depends on the Pak-interacting Rac-GEF PIX. In control cells, endogenous activated Pak and PIX translocate from focal complexes to cell-cell contacts during wound closure. This process is abrogated in cells expressing Pak1 kinase mutants. In contrast, Pak1 mutants rendered defective in PIX binding do not impede translocation of activated Pak and PIX, and exhibit normal wound healing. Thus, recruitment of activated Pak and PIX to cell-cell contacts is pivotal to transduction of growth-inhibitory signals from neighboring cells in epithelial wound healing.