RESUMO
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.
Assuntos
Polaridade Celular/fisiologia , Células Epiteliais/citologia , Imageamento Tridimensional/métodos , Animais , Técnicas de Cultura de Células/métodos , Linhagem Celular , Membrana Celular/química , Membrana Celular/ultraestrutura , Endossomos/química , Endossomos/fisiologia , Células Epiteliais/química , Células Epiteliais/fisiologia , Galactosiltransferases/análise , Proteínas da Matriz do Complexo de Golgi , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Membrana/análise , Microscopia Confocal , Microscopia Eletrônica , Microscopia de Fluorescência , Transportadores de Ânions Orgânicos Dependentes de Sódio/análise , Transportadores de Ânions Orgânicos Dependentes de Sódio/genética , Fosfoproteínas/análise , Compostos de Piridínio/metabolismo , Proteínas Qa-SNARE/análise , Proteínas Qa-SNARE/genética , Compostos de Amônio Quaternário/metabolismo , Proteínas R-SNARE/análise , Proteínas R-SNARE/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Simportadores/análise , Simportadores/genética , Junções Íntimas/química , Junções Íntimas/fisiologia , Transfecção , Tripsina/metabolismo , Proteína da Zônula de Oclusão-1RESUMO
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.