RESUMO
The correct targeting and trafficking of the adherens junction protein epithelial cadherin (E-cadherin) is a major determinant for the acquisition of epithelial cell polarity and for the maintenance of epithelial integrity. The compartments and trafficking components required to sort and transport E-cadherin to the basolateral cell surface remain to be fully defined. On the basis of previous data, we know that E-cadherin is trafficked via the recycling endosome (RE) in nonpolarized and newly polarized cells. Here we explore the role of the RE throughout epithelial morphogenesis in MDCK monolayers and cysts. Time-lapse microscopy in live cells, altering RE function biochemically, and expressing a dominant-negative form of Rab11 (DN-Rab11), each showed that the RE is always requisite for E-cadherin sorting and trafficking. The RE remained important for E-cadherin trafficking in MDCK cells from a nonpolarized state through to fully formed, polarized epithelial monolayers. During the development of epithelial cysts, DN-Rab11 disrupted E-cadherin targeting and trafficking, the subapical localization of pERM and actin, and cyst lumen formation. This final effect demonstrated an early and critical interdependence of Rab11 and the RE for E-cadherin targeting, apical membrane formation, and cell polarity in cysts.
Assuntos
Caderinas/metabolismo , Endossomos/metabolismo , Células Epiteliais/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Caderinas/genética , Linhagem Celular , Polaridade Celular/fisiologia , Cistos/metabolismo , Cistos/patologia , Cães , Endossomos/fisiologia , Células Epiteliais/citologia , Epitélio/crescimento & desenvolvimento , Epitélio/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Microscopia Confocal , Morfogênese/fisiologia , Mutação , Transporte Proteico/fisiologia , Receptores da Transferrina/genética , Receptores da Transferrina/fisiologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , Transferrina/metabolismo , Proteínas rab de Ligação ao GTP/genéticaRESUMO
E-cadherin is a major cell-cell adhesion protein of epithelia that is trafficked to the basolateral cell surface in a polarized fashion. The exact post-Golgi route and regulation of E-cadherin transport have not been fully described. The Rho GTPases Cdc42 and Rac1 have been implicated in many cell functions, including the exocytic trafficking of other proteins in polarized epithelial cells. These Rho family proteins are also associated with the cadherin-catenin complexes at the cell surface. We have used functional mutants of Rac1 and Cdc42 and inactivating toxins to demonstrate specific roles for both Cdc42 and Rac1 in the post-Golgi transport of E-cadherin. Dominant-negative mutants of Cdc42 and Rac1 accumulate E-cadherin at a distinct post-Golgi step. This accumulation occurs before p120(ctn) interacts with E-cadherin, because p120(ctn) localization was not affected by the Cdc42 or Rac1 mutants. Moreover, the GTPase mutants had no effect on the trafficking of a targeting mutant of E-cadherin, consistent with the selective involvement of Cdc42 and Rac1 in basolateral trafficking. These results provide a new example of Rho GTPase regulation of basolateral trafficking and demonstrate novel roles for Cdc42 and Rac1 in the post-Golgi transport of E-cadherin.
Assuntos
Caderinas/metabolismo , Proteína cdc42 de Ligação ao GTP/fisiologia , Proteínas rac1 de Ligação ao GTP/fisiologia , Animais , Células CHO , Linhagem Celular , Cricetinae , Cricetulus , Cães , Expressão Gênica , Rim/metabolismo , Mutação , Transporte Proteico/fisiologiaRESUMO
A key function of activated macrophages is to secrete proinflammatory cytokines such as TNFalpha; however, the intracellular pathway and machinery responsible for cytokine trafficking and secretion is largely undefined. Here we show that individual SNARE proteins involved in vesicle docking and fusion are regulated at both gene and protein expression upon stimulation with the bacterial cell wall component lipopolysaccharide. Focusing on two intracellular SNARE proteins, Vti1b and syntaxin 6 (Stx6), we show that they are up-regulated in conjunction with increasing cytokine secretion in activated macrophages and that their levels are selectively titrated to accommodate the volume and timing of post-Golgi cytokine trafficking. In macrophages, Vti1b and syntaxin 6 are localized on intracellular membranes and are present on isolated Golgi membranes and on Golgi-derived TNFalpha vesicles budded in vitro. By immunoprecipitation, we find that Vti1b and syntaxin 6 interact to form a novel intracellular Q-SNARE complex. Functional studies using overexpression of full-length and truncated proteins show that both Vti1b and syntaxin 6 function and have rate-limiting roles in TNFalpha trafficking and secretion. This study shows how macrophages have uniquely adapted a novel Golgi-associated SNARE complex to accommodate their requirement for increased cytokine secretion.
Assuntos
Proteínas de Transporte/metabolismo , Macrófagos/metabolismo , Proteínas de Membrana/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Regulação para Cima , Animais , Proteínas de Transporte/química , Linhagem Celular , Citocinas/metabolismo , Exocitose , Complexo de Golgi/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Immunoblotting , Imunoprecipitação , Inflamação , Lipopolissacarídeos/metabolismo , Proteínas de Membrana/química , Camundongos , Microscopia de Fluorescência , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Qa-SNARE , Proteínas Qb-SNARE , RNA/metabolismo , Proteínas SNARE , Fatores de Tempo , Proteínas de Transporte Vesicular/metabolismoRESUMO
Fibroblast growth factor (FGF) receptors (FGFRs) signal to modulate diverse cellular functions, including epithelial cell morphogenesis. In epithelial cells, E-cadherin plays a key role in cell-cell adhesion, and its function can be regulated through endocytic trafficking. In this study, we investigated the location, trafficking, and function of FGFR1 and E-cadherin and report a novel mechanism, based on endocytic trafficking, for the coregulation of E-cadherin and signaling from FGFR1. FGF induces the internalization of surface FGFR1 and surface E-cadherin, followed by nuclear translocation of FGFR1. The internalization of both proteins is regulated by common endocytic machinery, resulting in cointernalization of FGFR1 and E-cadherin into early endosomes. By blocking endocytosis, we show that this is a requisite, initial step for the nuclear translocation of FGFR1. Overexpression of E-cadherin blocks both the coendocytosis of E-cadherin and FGFR1, the nuclear translocation of FGFR1 and FGF-induced signaling to the mitogen-activated protein kinase pathway. Furthermore, stabilization of surface adhesive E-cadherin, by overexpressing p120ctn, also blocks internalization and nuclear translocation of FGFR1. These data reveal that conjoint endocytosis and trafficking is a novel mechanism for the coregulation of E-cadherin and FGFR1 during cell signaling and morphogenesis.
Assuntos
Transporte Ativo do Núcleo Celular , Caderinas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Cateninas , Adesão Celular , Moléculas de Adesão Celular/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , DNA Complementar/metabolismo , Eletroforese em Gel de Poliacrilamida , Endocitose , Endossomos/metabolismo , Células Epiteliais/metabolismo , Humanos , Immunoblotting , Sistema de Sinalização das MAP Quinases , Microscopia de Fluorescência , Fosfoproteínas/metabolismo , Plasmídeos/metabolismo , Transporte Proteico , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos , Transdução de Sinais , Fatores de Tempo , Transfecção , delta CateninaRESUMO
N4WBP5A (Ndfip2) belongs to an evolutionarily conserved group of Nedd4-interacting proteins with two homologues in mammalian species. We have previously shown that N4WBP5A expression in Xenopus oocytes results in increased cell-surface expression of the epithelial sodium channel. N4WBPs are characterized by one or two amino terminal PPxY motifs and three transmembrane domains. Here we show that both PPxY motifs of N4WBP5A mediate interaction with WW domains of Nedd4 and that N4WBP5A can physically interact with the WW domains of several Nedd4-family proteins. N4WBP5A is ubiquitinated and ubiquitination does not significantly affect the turnover of N4WBP5A protein. Ubiquitination of N4WBP5A is enhanced by Nedd4 and Nedd4-2 expression. N4WBP5A localizes to the Golgi, vesicles associated with the Golgi complex and to multivesicular bodies. We show that the ectopic expression of N4WBP5A inhibits receptor-mediated endocytosis of labelled epidermal growth factor. N4WBP5A overexpression inhibits accumulation of EGF in large endocytic/lysosomal vesicles suggestive of a role for N4WBP5A in protein trafficking. We propose that N4WBP5A acts as an adaptor to recruit Nedd4 family ubiquitin-protein ligases to the protein trafficking machinery.
Assuntos
Proteínas de Transporte/metabolismo , Endocitose/fisiologia , Complexo de Golgi/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas de Transporte/fisiologia , Linhagem Celular , Humanos , Imuno-Histoquímica , Proteínas de Membrana , Microscopia Eletrônica , Transporte ProteicoRESUMO
Galpha interacting protein (GAIP) is a regulator of G protein signaling protein that associates dynamically with vesicles and has been implicated in membrane trafficking, although its specific role is not yet known. Using an in vitro budding assay, we show that GAIP is recruited to a specific population of trans-Golgi network-derived vesicles and that these are distinct from coatomer or clathrin-coated vesicles. A truncation mutant (NT-GAIP) encoding only the N-terminal half of GAIP is recruited to trans-Golgi network membranes during the formation of vesicle carriers. Overexpression of NT-GAIP induces the formation of long, coated tubules, which are stabilized by microtubules. Results from the budding assay and from imaging in live cells show that these tubules remain attached to the Golgi stack rather than being released as carrier vesicles. NT-GAIP expression blocks membrane budding and results in the accumulation of tubular carrier intermediates. NT-GAIP-decorated tubules are competent to load vesicular stomatitis virus protein G-green fluorescent protein as post-Golgi, exocytic cargo and in cells expressing NT-GAIP there is reduced surface delivery of vesicular stomatitis virus protein G-green fluorescent protein. We conclude that GAIP functions as an essential part of the membrane budding machinery for a subset of post-Golgi exocytic carriers derived from the trans-Golgi network.
Assuntos
Proteínas Ativadoras de GTPase/metabolismo , Rede trans-Golgi/fisiologia , Rede trans-Golgi/ultraestrutura , Animais , Fracionamento Celular , Proteínas Ativadoras de GTPase/genética , Proteínas de Fluorescência Verde , Células HeLa , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia de Vídeo , Mutagênese , Coelhos , Proteínas Recombinantes de Fusão/metabolismo , Deleção de SequênciaRESUMO
Activation of macrophages with lipopolysaccharide (LPS) induces the rapid synthesis and secretion of proinflammatory cytokines, such as tumor necrosis factor (TNFalpha), for priming the immune response. TNFalpha plays a key role in inflammatory disease; yet, little is known of the intracellular trafficking events leading to its secretion. In order to identify molecules involved in this secretory pathway, we asked whether any of the known trafficking proteins are regulated by LPS. We found that the levels of SNARE proteins were rapidly and significantly up- or downregulated during macrophage activation. A subset of t-SNAREs (Syntaxin 4/SNAP23/Munc18c) known to control regulated exocytosis in other cell types was substantially increased by LPS in a temporal pattern coinciding with peak TNFalpha secretion. Syntaxin 4 formed a complex with Munc18c at the cell surface of macrophages. Functional studies involving the introduction of Syntaxin 4 cDNA or peptides into macrophages implicate this t-SNARE in a rate-limiting step of TNFalpha secretion and in membrane ruffling during macrophage activation. We conclude that, in macrophages, SNAREs are regulated in order to accommodate the rapid onset of cytokine secretion and for membrane traffic associated with the phenotypic changes of immune activation. This represents a novel regulatory role for SNAREs in regulated secretion and in macrophage-mediated host defense.