title: Four-dimensional imaging of filter-grown polarized epithelial cells.
abstract: 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.
authors.affiliation: Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA. wakabayo@mail.nih.gov
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Wakabayashi
authors.forename: Jennifer
authors.initials: J
authors.lastname: Chua
authors.forename: Janet M
authors.initials: JM
authors.lastname: Larkin
authors.forename: Jennifer
authors.initials: J
authors.lastname: Lippincott-Schwartz
authors.forename: Irwin M
authors.initials: IM
authors.lastname: Arias
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry / Q000648:ultrastructure; D016764:Cell Polarity / Q000502:physiology*; D011992:Endosomes / Q000737:chemistry / Q000502:physiology; D004847:Epithelial Cells / Q000737:chemistry / Q000166:cytology* / Q000502:physiology; D005700:Galactosyltransferases / Q000032:analysis; D000077614:Golgi Matrix Proteins; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D021621:Imaging, Three-Dimensional / Q000379:methods*; D008565:Membrane Proteins / Q000032:analysis; D018613:Microscopy, Confocal; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D029363:Organic Anion Transporters, Sodium-Dependent / Q000032:analysis / Q000235:genetics; D010750:Phosphoproteins / Q000032:analysis; D011726:Pyridinium Compounds / Q000378:metabolism; D050765:Qa-SNARE Proteins / Q000032:analysis / Q000235:genetics; D000644:Quaternary Ammonium Compounds / Q000378:metabolism; D050683:R-SNARE Proteins / Q000032:analysis / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D027981:Symporters / Q000032:analysis / Q000235:genetics; D019108:Tight Junctions / Q000737:chemistry / Q000502:physiology; D014162:Transfection; D014357:Trypsin / Q000378:metabolism; D062826:Zonula Occludens-1 Protein
chemical_list: C092350:FM 4-64; D000077614:Golgi Matrix Proteins; D008565:Membrane Proteins; D029363:Organic Anion Transporters, Sodium-Dependent; D010750:Phosphoproteins; D011726:Pyridinium Compounds; D050765:Qa-SNARE Proteins; D000644:Quaternary Ammonium Compounds; D050683:R-SNARE Proteins; D011993:Recombinant Fusion Proteins; D027981:Symporters; C569042:TJP1 protein, human; D062826:Zonula Occludens-1 Protein; C087574:macrogolgin; C071908:sodium-bile acid cotransporter; D049452:Green Fluorescent Proteins; D005700:Galactosyltransferases; D014357:Trypsin