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© The Rockefeller University Press, 0021-9525/1999//645 $5.00
The Journal of Cell Biology, Volume 146, Number 3, , 1999 645-658

Original Article

Insoluble {gamma}-Tubulin–Containing Structures Are Anchored to the Apical Network of Intermediate Filaments in Polarized Caco-2 Epithelial Cells



Pedro J.I. Salasa

a University of Miami School of Medicine, Department of Cell Biology and Anatomy, Miami, Florida 33101
University of Miami School of Medicine, Department of Cell Biology and Anatomy, R-124, P.O. Box 016960, Miami, FL 33101.(305) 545-7166(305) 243-6977

psalas{at}mednet.med.miami.edu

We have previously shown that a thin (~1 µm) layer of intermediate filaments located beneath the apical membrane of a variety of simple epithelial cells participates in the organization of apical microfilaments and microtubules. Here, I confirmed the apical distribution of {gamma}-tubulin–containing structures (potential microtubule-organizing centers) in CACO-2 cells and demonstrated perfect colocalization of centrosomes and nearly 50% of noncentrosomal {gamma}-tubulin with apical intermediate filaments, but not with apical F-actin. Furthermore, the antisense-oligonucleotide–mediated downregulation of cytokeratin 19, using two different antisense sequences, was more efficient than anticytoskeletal agents to delocalize centrosomes. Electron microscopy colocalization suggests that binding occurs at the outer boundary of the pericentriolar material. Type I cytokeratins 18 and 19 present in these cells specifically coimmunoprecipitated in multi-protein fragments of the cytoskeleton with {gamma}-tubulin. The size and shape of the fragments, visualized at the EM level, indicate that physical trapping is an unlikely explanation for this result. Drastic changes in the extraction protocol did not affect coimmunoprecipitation. These results from three independent techniques, indicate that insoluble {gamma}-tubulin–containing structures are attached to apical intermediate filaments.

Key Words: cell polarity • centrosome • {gamma}-tubulin • intermediate filaments • keratin

© 1999 The Rockefeller University Press

1.used in this paper: CK, cytokeratin; EB, extraction buffer (PBS, 2 mM EDTA, 1% Triton X-100); IF, intermediate filaments; i.p., immunoprecipitation; KEB, extraction buffer (70 mM KCl, 80 mM Pipes, pH 6.5, 5 mM EGTA, 2 mM MgCl2, 0.1% saponin); MTOC, microtubule-organizing center


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