Preservation of structural and functional polarity in isolated epithelial cells.
We describe a method to isolate epithelial cells from gallbladders of Necturus maculosus with preserved structural and functional polarity. Isolation was carried out with a mixture of collagenase and protease, with only a brief exposure to a divalent-cation-free medium. About 40% of the isolated epithelial cells had a "figure-eight" shape and retained metabolic and cell membrane integrity. Figure-eight cells display features consistent with preserved polarity for several hours, including the following: 1) the "apical" and "basolateral" membrane domains were differentially labeled by a hydrophobic fluorescent dye; 2) freeze fracture electron microscopy verified two plasma membrane domains differing in the presence of microvilli and folds and separated by tight junctions; 3) proteins such as ZO-1, NHE3, and Na(+)-K(+)-ATPase remained localized in the junctional, apical, and basolateral regions, respectively; 4) after apical surface exposure to wheat germ agglutinin, the label remained in the apical membrane after cell isolation; and 5) patch-clamp experiments demonstrated polarized expression of K+ channels. Polarity was rapidly lost after removal of extracellular Ca2+, exposure to trypsin, or ATP depletion. Therefore, this preparation allows for structural and functional studies of epithelial transport in single cells retaining the essential features present in the assembled epithelium.
Torres
R J
RJ
Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston 77555, USA.
Altenberg
G A
GA
Copello
J A
JA
Zampighi
G
G
Reuss
L
L
D000818:Animals; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism; D016764:Cell Polarity; D002470:Cell Survival; D003584:Cytological Techniques; D004847:Epithelial Cells; D004848:Epithelium / Q000502:physiology / Q000648:ultrastructure; D005704:Gallbladder / Q000166:cytology / Q000502:physiology* / Q000648:ultrastructure; D066298:In Vitro Techniques; D007425:Intracellular Membranes / Q000378:metabolism; D007536:Isomerism; D008854:Microscopy, Electron; D009338:Necturus maculosus; D018408:Patch-Clamp Techniques; D015221:Potassium Channels / Q000737:chemistry / Q000378:metabolism
D015221:Potassium Channels