VIMENTIN FILAMENTS FOLLOW THE PREEXISTING CYTOKERATIN NETWORK DURING EPITHELIAL-MESENCHYMAL TRANSITION OF CULTURED NEONATAL RAT HEPATOCYTES

Changes in cell cytoskeleton are known to play an important role in di fferentiation and embryogenesis and also in carcinogenesis. Previous s tudies indicated that neonatal hepatocytes undergo an epithelial-mesen chymal transition when cultured in a serum-free medium for several day s. Here we show by Western blotting of neonatal rat liver cells cultur ed for 3 days that vimentin and cytokeratin were expressed by these ce lls. Epidermal growth factor treatment induced high coexpression of vi mentin and cytokeratin filaments in hepatocytes from neonatal livers, as detected by double immunofluorescence microscopy. Confocal scanning laser microscopy was used to determine the spatial and cell distribut ion of cytokeratin and vimentin intermediate filament networks, Viment in-expressing hepatocytes were mainly located on the periphery of epit helial clusters and presented a migratory morphology, suggesting that vimentin expression was related to the loss of cell-cell contact. Shor t vimentin filaments were mainly located at the cytoplasmic sites behi nd the extending lamella. Horizontal and vertical dual imaging of doub le immunofluorescence with antivimentin and anti-cytokeratin antibodie s indicated that both filaments colocalize strongly. Three-dimensional reconstruction of serial optical sections revealed that newly synthes ized vimentin distributed following the preexisting cytokeratin networ k and, when present, both filament scaffolds codistributed inside cult ured hepatocytes. Immunoelectron microscopy performed in whole-mount-e xtracted cultured cells revealed that both filaments are closely inter related but independent. However, a high degree of immunogold colocali zation was found in the knots of the filament network. Further experim ents with colcemide and cytochalasin treatment indicated that vimentin filament distribution, but not cytokeratin, was dependent on an intac t microtubule network. These results are consistent with a mechanism o f vimentin assembly, whereby growth of vimentin intermediate filaments is dependent on microtubules in topographically restricted cytoplasmi c sites, in close relation to the cytokeratin cytoskeleton and to chan ges in cell-cell contact and cell shape. (C) 1996 Academic Press, Inc.