RESUMEN
Epsin is an evolutionarily conserved endocytic clathrin adaptor whose most critical function(s) in clathrin coat dynamics remain(s) elusive. To elucidate such function(s), we generated embryonic fibroblasts from conditional epsin triple KO mice. Triple KO cells displayed a dramatic cell division defect. Additionally, a robust impairment in clathrin-mediated endocytosis was observed, with an accumulation of early and U-shaped pits. This defect correlated with a perturbation of the coupling between the clathrin coat and the actin cytoskeleton, which we confirmed in a cell-free assay of endocytosis. Our results indicate that a key evolutionary conserved function of epsin, in addition to other roles that include, as we show here, a low affinity interaction with SNAREs, is to help generate the force that leads to invagination and then fission of clathrin-coated pits.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/genética , Clatrina/metabolismo , Invaginaciones Cubiertas de la Membrana Celular/metabolismo , Endocitosis/genética , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/ultraestructura , Actinas/genética , Proteínas Adaptadoras del Transporte Vesicular/deficiencia , Animales , Clatrina/genética , Invaginaciones Cubiertas de la Membrana Celular/genética , Invaginaciones Cubiertas de la Membrana Celular/ultraestructura , Embrión de Mamíferos , Fibroblastos/metabolismo , Fibroblastos/ultraestructura , Expresión Génica , Ratones , Ratones Noqueados , Cultivo Primario de Células , Unión Proteica , Proteínas SNARE/genética , Proteínas SNARE/metabolismo , Transducción de SeñalRESUMEN
Epsins are a family of ubiquitin-binding, endocytic clathrin adaptors. Mice lacking both epsins 1 and 2 (Epn1/2) die at embryonic day 10 and exhibit an abnormal vascular phenotype. To examine the angiogenic role of endothelial epsins, we generated mice with constitutive or inducible deletion of Epn1/2 in vascular endothelium. These mice exhibited no abnormal phenotypes under normal conditions, suggesting that lack of endothelial epsins 1 and 2 did not affect normal blood vessels. In tumors, however, loss of epsins 1 and 2 resulted in disorganized vasculature, significantly increased vascular permeability, and markedly retarded tumor growth. Mechanistically, we show that VEGF promoted binding of epsin to ubiquitinated VEGFR2. Loss of epsins 1 and 2 specifically impaired endocytosis and degradation of VEGFR2, which resulted in excessive VEGF signaling that compromised tumor vascular function by exacerbating nonproductive leaky angiogenesis. This suggests that tumor vasculature requires a balance in VEGF signaling to provide sufficient productive angiogenesis for tumor development and that endothelial epsins 1 and 2 negatively regulate the output of VEGF signaling. Promotion of excessive VEGF signaling within tumors via a block of epsin 1 and 2 function may represent a strategy to prevent normal angiogenesis in cancer patients who are resistant to anti-VEGF therapies.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/genética , Proliferación Celular , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular/fisiología , Proteínas Adaptadoras del Transporte Vesicular/deficiencia , Animales , Permeabilidad Capilar , Carcinoma Pulmonar de Lewis/irrigación sanguínea , Carcinoma Pulmonar de Lewis/metabolismo , Carcinoma Pulmonar de Lewis/patología , Movimiento Celular , Endocitosis , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana/fisiología , Humanos , Uniones Intercelulares/metabolismo , Uniones Intercelulares/patología , Masculino , Ratones , Ratones Noqueados , Trasplante de Neoplasias , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Proteolisis , Carga Tumoral , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Epsin is a ubiquitin-binding endocytic adaptor, which is highly concentrated at clathrin-coated pits and coordinates acquisition of bilayer curvature with coat recruitment and cargo selection. Epsin is encoded by three distinct genes in mammals. Epsin 1 and 2 have broad tissue distribution with high-level expression in the brain. In contrast, epsin 3 was reported to be expressed primarily in immature keratinocytes. Here, we show that epsin 3 is selectively expressed at high levels in the stomach (including the majority of gastric cancers), where it is concentrated in parietal cells. In these cells, epsin 3 is enriched and colocalized with clathrin around apical canaliculi, the sites that control acidification of the stomach lumen via the exo-endocytosis of vesicles containing the H/K ATPase. Deletion of the epsin 3 gene in mice did not result in obvious pathological phenotypes in either the stomach or other organs, possibly because of overlapping functions of the other two epsins. However, levels of EHD1 and EHD2, two membrane tubulating proteins with a role in endocytic recycling, were elevated in epsin 3 knock-out stomachs, pointing to a functional interplay of epsin 3 with EHD proteins in the endocytic pathway of parietal cells. We suggest that epsin 3 cooperates with other bilayer binding proteins with curvature sensing/generating properties in the specialized traffic and membrane remodeling processes typical of gastric parietal cells.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Polaridad Celular , Endocitosis/fisiología , Mucosa Gástrica/metabolismo , Células Parietales Gástricas/fisiología , Proteínas Adaptadoras del Transporte Vesicular/genética , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , ATPasa Intercambiadora de Hidrógeno-Potásio/metabolismo , Humanos , Ratones , Ratones Noqueados , Células Parietales Gástricas/ultraestructura , Estómago/anatomía & histología , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patología , Distribución Tisular , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMEN
Epsins are endocytic adaptors with putative functions in general aspects of clathrin-mediated endocytosis as well as in the internalization of specific membrane proteins. We have now tested the role of the ubiquitously expressed epsin genes, Epn1 and Epn2, by a genetic approach in mice. While either gene is dispensable for life, their combined inactivation results in embryonic lethality at E9.5-E10, i.e., at the beginning of organogenesis. Consistent with studies in Drosophila, where epsin endocytic function was linked to Notch activation, developmental defects observed in epsin 1/2 double knockout (DKO) embryos recapitulated those produced by a global impairment of Notch signaling. Accordingly, expression of Notch primary target genes was severely reduced in DKO embryos. However, housekeeping forms of clathrin-mediated endocytosis were not impaired in cells derived from these embryos. These findings support a role of epsin as a specialized endocytic adaptor, with a critical role in the activation of Notch signaling in mammals.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/fisiología , Regulación del Desarrollo de la Expresión Génica , Receptores Notch/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Animales , Clatrina/metabolismo , Endocitosis , Fibroblastos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Genéticos , Fenotipo , Transducción de Señal , Factores de Tiempo , Distribución TisularRESUMEN
The GTPase dynamin, a key player in endocytic membrane fission, interacts with numerous proteins that regulate actin dynamics and generate/sense membrane curvature. To determine the functional relationship between these proteins and dynamin, we have analyzed endocytic intermediates that accumulate in cells that lack dynamin (derived from dynamin 1 and 2 double conditional knockout mice). In these cells, actin-nucleating proteins, actin, and BAR domain proteins accumulate at the base of arrested endocytic clathrin-coated pits, where they support the growth of dynamic long tubular necks. These results, which we show reflect the sequence of events in wild-type cells, demonstrate a concerted action of these proteins prior to, and independent of, dynamin and emphasize similarities between clathrin-mediated endocytosis in yeast and higher eukaryotes. Our data also demonstrate that the relationship between dynamin and actin is intimately connected to dynamin's endocytic role and that dynamin terminates a powerful actin- and BAR protein-dependent tubulating activity.