Insoluble gamma-tubulin-containing structures are anchored to the apical network of intermediate filaments in polarized CACO-2 epithelial cells

We have previously shown that a thin (similar to 1 mu m) layer of intermedi ate filaments located beneath the apical membrane of a variety of simple ep ithelial cells participates in the organization of apical microfilaments an d 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 no t 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 oute r boundary of the pericentriolar material. Type I cytokeratins 18 and 19 pr esent in these cells specifically coimmunoprecipitated in multi-protein fra gments of the cytoskeleton with gamma-tubulin, The size and shape of the fr agments, visualized at the EM level, indicate that physical trapping is an unlikely explanation for this result, Drastic changes in the extraction pro tocol did not affect coimmunoprecipitation. These results from three indepe ndent techniques, indicate that insoluble gamma-tubulin-containing structur es are attached to apical intermediate filaments.