RESUMEN
Understanding how epithelial cells generate and maintain polarity and function requires live cell imaging. In order for cells to become fully polarized, it is necessary to grow them on a permeable membrane filter; however, the translucent filter obstructs the microscope light path required for quantitative live cell imaging. Alternatively, the membrane filter may be excised but this eliminates selective access to apical and basolateral surfaces. Conversely, epithelial cells cultured directly on glass exhibit different phenotypes and functions from filter grown cells. Here, we describe a new method for culturing polarized epithelial cells on a Transwell filter insert that allows superior live cell imaging with spatial and temporal image resolution previously unachievable using conventional methods. Cells were cultured on the underside of a filter support. Epithelial cells grown in this inverted configuration exhibit a fully polarized architecture, including the presence of functional tight junctions. This new culturing system permits four-dimensional (three spatial dimension over time) imaging of endosome and Golgi apparatus dynamics, and permits selective manipulation of the apical and basolateral surfaces. This new technique has wide applicability for visualization and manipulation of polarized epithelial cells.
Asunto(s)
Polaridad Celular/fisiología , Células Epiteliales/citología , Imagenología Tridimensional/métodos , Animales , Técnicas de Cultivo de Célula/métodos , Línea Celular , Membrana Celular/química , Membrana Celular/ultraestructura , Endosomas/química , Endosomas/fisiología , Células Epiteliales/química , Células Epiteliales/fisiología , Galactosiltransferasas/análisis , Proteínas de la Matriz de Golgi , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas de la Membrana/análisis , Microscopía Confocal , Microscopía Electrónica , Microscopía Fluorescente , Transportadores de Anión Orgánico Sodio-Dependiente/análisis , Transportadores de Anión Orgánico Sodio-Dependiente/genética , Fosfoproteínas/análisis , Compuestos de Piridinio/metabolismo , Proteínas Qa-SNARE/análisis , Proteínas Qa-SNARE/genética , Compuestos de Amonio Cuaternario/metabolismo , Proteínas R-SNARE/análisis , Proteínas R-SNARE/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Simportadores/análisis , Simportadores/genética , Uniones Estrechas/química , Uniones Estrechas/fisiología , Transfección , Tripsina/metabolismo , Proteína de la Zonula Occludens-1RESUMEN
Bile duct ligation (BDL) impairs basolateral-to-apical transcytosis in hepatocytes, causing accumulation of transcytotic carriers for the polymeric IgA receptor (pIgA-R) and redistribution of secretory component (SC) from bile to blood. To gain insight into the mechanisms regulating transcytosis and the pathophysiology of cholestasis, we investigated nascent protein trafficking in control and BDL livers using cell fractionation in the context of in vivo pulse-chase experiments and immunoblot analysis. Control and cholestatic hepatocytes trafficked [35S]-labeled serum proteins and the pIgA-R along the secretory pathway with identical kinetics. However, BDL impaired transcytosis, causing (1) accumulation of the pIgA-R, rab3D, rab11a, and other candidate regulators of apical-directed secretion in a crude vesicle carrier fraction (CVCF) enriched in transcytotic carriers; (2) slow delivery of [35S]-labeled SC to bile; and (3) paracellular reflux of SC from bile to blood. In conclusion, these data indicate that the secretory and transcytotic pathways remain polarized in cholestatic hepatocytes and suggest that the pIgA-R traffics through postendosomal rab3D-, rab11a-, and syntaxin 2-associated compartments, implicating these proteins in the regulation of transcytosis.
