RESUMEN
BACKGROUND & AIMS: In epithelial cells, protein sorting mechanisms regulate localization of plasma membrane proteins that generate and maintain cell polarity. The clathrin-adaptor protein (AP) complex AP-1B is expressed specifically in polarized epithelial cells, where it regulates basolateral sorting of membrane proteins. However, little is known about its physiological significance. METHODS: We analyzed the intestinal epithelia of mice deficient in Ap1m2 (Ap1m2(-/-) mice), which encodes the AP-1B µ1B subunit, and compared it with 129/B6/CD1 littermates (controls). Notch signaling was inhibited by intraperitoneal injection of dibenzazepine, and ß-catenin signaling was inhibited by injection of IWR1. Intestinal tissue samples were collected and analyzed by immunofluorescence analysis. RESULTS: Ap1m2(-/-) mice developed intestinal epithelial cell hyperplasia. The polarity of intestinal epithelial cells was disrupted, as indicated by the appearance of ectopic microvilli-like structures on the lateral plasma membrane and mislocalization of basolateral membrane proteins, including the low-density lipoprotein receptor and E-cadherin. The E-cadherin-ß-catenin complex therefore was disrupted at the adherens junction, resulting in nuclear translocation of ß-catenin. This resulted in up-regulation of genes regulated by ß-catenin/transcription factor 4 (Tcf4) complex, and increased the proliferation of intestinal epithelial cells. CONCLUSIONS: AP-1B is required for protein sorting and polarization of intestinal cells in mice. Loss of AP-1B in the intestinal epithelia results in mislocalization of E-cadherin, activation of ß-catenin/Tcf4 complex, proliferation, and hyperplasia.
Asunto(s)
Complejo 1 de Proteína Adaptadora/deficiencia , Subunidades mu de Complejo de Proteína Adaptadora/deficiencia , Polaridad Celular , Proliferación Celular , Células Epiteliales/metabolismo , Mucosa Intestinal/metabolismo , Intestino Delgado/metabolismo , Complejo 1 de Proteína Adaptadora/fisiología , Subunidades mu de Complejo de Proteína Adaptadora/fisiología , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Biomarcadores/metabolismo , Cadherinas/metabolismo , Células Epiteliales/patología , Células Epiteliales/fisiología , Femenino , Técnica del Anticuerpo Fluorescente , Mucosa Intestinal/patología , Mucosa Intestinal/fisiopatología , Intestino Delgado/patología , Intestino Delgado/fisiopatología , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Noqueados , Factor de Transcripción 4 , beta Catenina/metabolismoRESUMEN
Dictyostelium discoideum cells continuously internalize extracellular material, which accumulates in well-characterized endocytic vacuoles. In this study, we describe a new endocytic compartment identified by the presence of a specific marker, the p25 protein. This compartment presents features reminiscent of mammalian recycling endosomes: it is localized in the pericentrosomal region but distinct from the Golgi apparatus. It specifically contains surface proteins that are continuously endocytosed but rapidly recycled to the cell surface and thus absent from maturing endocytic compartments. We evaluated the importance of each clathrin-associated adaptor complex in establishing a compartmentalized endocytic system by studying the phenotype of the corresponding mutants. In knockout cells for mu3, a subunit of the AP-3 clathrin-associated complex, membrane proteins normally restricted to p25-positive endosomes were mislocalized to late endocytic compartments. Our results suggest that AP-3 plays an essential role in the compartmentalization of the endocytic pathway in Dictyostelium.
Asunto(s)
Complejo 3 de Proteína Adaptadora/fisiología , Dictyostelium/fisiología , Endocitosis/fisiología , Subunidades mu de Complejo de Proteína Adaptadora/deficiencia , Subunidades mu de Complejo de Proteína Adaptadora/genética , Proteínas Adaptadoras del Transporte Vesicular/genética , Animales , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Membrana Celular/metabolismo , Dictyostelium/genética , Endosomas/metabolismo , Eliminación de Gen , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/metabolismo , Modelos Biológicos , Transporte de Proteínas/fisiología , Proteínas Protozoarias/genética , Proteínas Protozoarias/inmunología , Proteínas Protozoarias/metabolismo , ATPasas de Translocación de Protón Vacuolares/metabolismoRESUMEN
Calcineurin is a highly conserved regulator of Ca(2+) signaling in eukaryotes. In fission yeast, calcineurin is not essential for viability but is required for cytokinesis and Cl(-) homeostasis. In a genetic screen for mutations that are synthetically lethal with calcineurin deletion, we isolated a mutant, cis1-1/apm1-1, an allele of the apm1(+) gene that encodes a homolog of the mammalian micro1A subunit of the clathrin-associated adaptor protein-1 (AP-1) complex. The cis1-1/apm1-1 mutant as well as the apm1-deleted (Deltaapm1) cells showed distinct phenotypes: temperature sensitivity; tacrolimus (FK506) sensitivity; and pleiotropic defects in cytokinesis, cell integrity, and vacuole fusion. Electron micrographs revealed that Deltaapm1 cells showed large vesicular structures associated with Golgi stacks and accumulated post-Golgi secretory vesicles. Deltaapm1 cells also showed the massive accumulation of the exocytic v-SNARE Syb1 in the Golgi/endosomes and a reduced secretion of acid phosphatase. These phenotypes observed in apm1 mutations were accentuated upon temperature up-shift and FK506 treatment. Notably, Apm1-GFP localized to the Golgi/endosomes, the spindle pole bodies, and the medial region. These findings suggest a role for Apm1 associated with the Golgi/endosome function, thereby affecting various cellular processes, including secretion, cytokinesis, vacuole fusion, and cell integrity and also suggest that calcineurin is involved in these events.