Synthesis and phosphorylation of cytoskeletal proteins during the in vitro biogenesis of MDCK cell monolayers

In vitro formation of a functional MDCK cell monolayer requires the dynamic participation of the cytoskeleton. Cell shape, contacts and polarity, as well as transepithelial electric resistance (TER), are actively modified during this differentiation process. We studied the distribution and rearrangement of cytokeratin, vimentin and actin filaments that occur in the monolayer concomitant with the synthesis and phosphorylation of these proteins. Cells cultured for short time in suspension, subconfluent or early confluent cells, showed striking differences in cell shape and arrangement of their cytoskeletal filaments. Subconfluent and early confluent cells synthesized proteins at high levels. In contrast, suspended cells maintained lower rates of protein synthesis. Labeled amino acid uptake was very similar in all these culture conditions. Gel electrophoresis analyses of the synthesized proteins showed increases in the synthesis of actin, vimentin and specific cytokeratins in subconfluent and early confluent cells. On the other hand a decrease in total protein, actin, vimentin and cytokeratin synthesis was observed in cells kept in suspension for 24 h or in 78-h or 174-h older confluent cultures. These cultures also showed a decrease in the uptake of labeled amino acid. Cytokeratin and vimentin phosphorylation rates were also modified during the in vitro formation of a monolayer. In suspended cells, cytokeratins were phosphorylated and three labeled isoelectric variants of the 40, 48 and 58K (K = 10(3) Mr) cytokeratins were present in intermediate filament extracts. In subconfluent and early confluent cells only two isoelectric phosphorylated variants of the 40, 48 and 58K cytokeratins were detected and vimentin was also phosphorylated.(ABSTRACT TRUNCATED AT 250 WORDS)