Advertisement

Four-dimensional imaging of filter-grown polarized epithelial cells

  • 1053 Accesses

  • 19 Citations

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.

This is a preview of subscription content, log in to check access.

Access options

Buy single article

Instant unlimited access to the full article PDF.

US$ 39.95

Price includes VAT for USA

Subscribe to journal

Immediate online access to all issues from 2019. Subscription will auto renew annually.

US$ 199

This is the net price. Taxes to be calculated in checkout.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

References

  1. Bacallao R, Antony C, Dotti C, Karsenti E, Stelzer EH, Simons K (1989) The subcellular organization of Madin-Darby canine kidney cells during the formation of a polarized epithelium. J Cell Biol 109:2817–2832

  2. Balcarova-Stander J, Pfeiffer SE, Fuller SD, Simons K (1984) Development of cell surface polarity in the epithelial Madin-Darby canine kidney (MDCK) cell line. EMBO J 3:2687–2694

  3. Betz WJ, Mao F, Smith CB (1996) Imaging exocytosis and endocytosis. Curr Opin Neurobiol 6:365–371

  4. Bomsel M, Prydz K, Parton RG, Gruenberg J, Simons K (1989) Endocytosis in filter-grown Madin-Darby canine kidney cells. J Cell Biol 109:3243–3258

  5. Cereijido M, Robbins ES, Dolan WJ, Rotunno CA, Sabatini DD (1978) Polarized monolayers formed by epithelial cells on a permeable and translucent support. J Cell Biol 77:853–880

  6. Duclos S, Diez R, Garin J, Papadopoulou B, Descoteaux A, Stenmark H, Desjardins M (2000) Rab5 regulates the kiss and run fusion between phagosomes and endosomes and the acquisition of phagosome leishmanicidal properties in RAW 264.7 macrophages. J Cell Sci 113 Pt 19:3531–3541

  7. Fuller S, von Bonsdorff CH, Simons K (1984) Vesicular stomatitis virus infects and matures only through the basolateral surface of the polarized epithelial cell line, MDCK. Cell 38:65–77

  8. Hua W, Sheff D, Toomre D, Mellman I (2006) Vectorial insertion of apical and basolateral membrane proteins in polarized epithelial cells revealed by quantitative 3D live cell imaging. J Cell Biol 172:1035–1044

  9. Kreitzer G, Marmorstein A, Okamoto P, Vallee R, Rodriguez-Boulan E (2000) Kinesin and dynamin are required for post-Golgi transport of a plasma-membrane protein. Nat Cell Biol 2:125–127

  10. Kreitzer G, Schmoranzer J, Low SH, Li X, Gan Y, Weimbs T, Simon SM, Rodriguez-Boulan E (2003) Three-dimensional analysis of post-Golgi carrier exocytosis in epithelial cells. Nat Cell Biol 5:126–136

  11. Low SH, Chapin SJ, Weimbs T, Komuves LG, Bennett MK, Mostov KE (1996) Differential localization of syntaxin isoforms in polarized Madin-Darby canine kidney cells. Mol Biol Cell 7:2007–2018

  12. Matter K, Mellman I (1994) Mechanisms of cell polarity: sorting and transport in epithelial cells. Curr Opin Cell Biol 6:545–554

  13. Misfeldt DS, Hamamoto ST, Pitelka DR (1976) Transepithelial transport in cell culture. Proc Natl Acad Sci USA 73:1212–1216

  14. Mostov K, Su T, ter Beest M (2003) Polarized epithelial membrane traffic: conservation and plasticity. Nat Cell Biol 5:287–293

  15. Nichols BJ, Kenworthy AK, Polishchuk RS, Lodge R, Roberts TH, Hirschberg K, Phair RD, Lippincott-Schwartz J (2001) Rapid cycling of lipid raft markers between the cell surface and Golgi complex. J Cell Biol 153:529–541

  16. Paladino S, Pocard T, Catino MA, Zurzolo C (2006) GPI-anchored proteins are directly targeted to the apical surface in fully polarized MDCK cells. J Cell Biol 172:1023–1034

  17. Presley JF, Cole NB, Schroer TA, Hirschberg K, Zaal KJ, Lippincott-Schwartz J (1997) ER-to-Golgi transport visualized in living cells. Nature 389:81–85

  18. Richardson JC, Scalera V, Simmons NL (1981) Identification of two strains of MDCK cells which resemble separate nephron tubule segments. Biochim Biophys Acta 673:26–36

  19. Richardson JC, Simmons NL (1979) Demonstration of protein asymmetries in the plasma membrane of cultured renal (MDCK) epithelial cells by lactoperoxidase-mediated iodination. FEBS Lett 105:201–204

  20. Rodriguez-Boulan E, Kreitzer G, Musch A (2005) Organization of vesicular trafficking in epithelia. Nat Rev Mol Cell Biol 6:233–247

  21. Rosenberg SO, Berkowitz PA, Li L, Schuster VL (1991) Imaging of filter-grown epithelial cells: MDCK Na(+)-H+ exchanger is basolateral. Am J Physiol 260:C868–C876

  22. Simons K, Fuller SD (1985) Cell surface polarity in epithelia. Annu Rev Cell Biol 1:243–288

  23. Steegmaier M, Lee KC, Prekeris R, Scheller RH (2000) SNARE protein trafficking in polarized MDCK cells. Traffic 1:553–560

  24. Sun AQ, Arrese MA, Zeng L, Swaby I, Zhou MM, Suchy FJ (2001) The rat liver Na(+)/bile acid cotransporter. Importance of the cytoplasmic tail to function and plasma membrane targeting. J Biol Chem 276:6825–6833

  25. von Bonsdorff CH, Fuller SD, Simons K (1985) Apical and basolateral endocytosis in Madin-Darby canine kidney (MDCK) cells grown on nitrocellulose filters. EMBO J 4:2781–2792

  26. Wakabayashi Y, Lippincott-Schwartz J, Arias IM (2004) Intracellular trafficking of bile salt export pump (ABCB11) in polarized hepatic cells: constitutive cycling between the canalicular membrane and rab11-positive endosomes. Mol Biol Cell 15:3485–3496

  27. Wang E, Brown PS, Aroeti B, Chapin SJ, Mostov KE, Dunn KW (2000) Apical and basolateral endocytic pathways of MDCK cells meet in acidic common endosomes distinct from a nearly-neutral apical recycling endosome. Traffic 1:480–493

Download references

Acknowledgments

We thank Vicrotia C. Cogger for assistance with SEM; Roberto Weigert (NHLBI, NIH, Bethesda, MD, USA), Frederick J. Suchy (Mount Sinai School of Medicine, New York, NY, USA) and Adam D. Linstedt (Carnegie Mellon University, Pittsburgh, PA, USA) for providing reagents used in this study. Janet L. Larkin received support from the Oak Ridge Institute for Science and Education.

Author information

Correspondence to Yoshiyuki Wakabayashi.

Electronic supplementary material

Movie 1 Z sections of VAMP 8-GFP positive endosomes in live MDCKmonolayer. (mov 2.87 MB)

Movie 2 Time lapse imaging of VAMP 8- GFP positive endosome inpolarized MDCK cells. (mov 2.85 MB)

Movie 3 Z sections of VAMP 8-GFP positive endosome in fixed MDCKmonolayer. (mov 2.77 MB)

Movie 4 4D imaging of Golgi fragmentation caused by BFA in polarizedMDCK cells. (mov 2.14 MB)

Movie 1 Z sections of VAMP 8-GFP positive endosomes in live MDCKmonolayer. (mov 2.87 MB)

Movie 2 Time lapse imaging of VAMP 8- GFP positive endosome inpolarized MDCK cells. (mov 2.85 MB)

Movie 3 Z sections of VAMP 8-GFP positive endosome in fixed MDCKmonolayer. (mov 2.77 MB)

Movie 4 4D imaging of Golgi fragmentation caused by BFA in polarizedMDCK cells. (mov 2.14 MB)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Wakabayashi, Y., Chua, J., Larkin, J.M. et al. Four-dimensional imaging of filter-grown polarized epithelial cells. Histochem Cell Biol 127, 463–472 (2007) doi:10.1007/s00418-007-0274-x

Download citation

Keywords

  • Four-dimensional imaging
  • Live cell imaging
  • Polarized epithelial cells
  • Permeable filter insert
  • MDCK cells