Translocation of MARCKS and reorgnization of the cytoskeleton by PMA correlates with the ion selectivity, the confluence, and transformation state ofkidney epithelial cell lines

The role of protein kinase C (PKC) in the regulation of the cytoskeleton of epithelial cells with lightly sealed contacts, poor contacts, and without contacts were investigated by incubating them with a protein kinase C activ ator phorbol myristoyl acetate (PMA). The morphology and organization of th e membrane skeleton and stress fibers as well as the localization of an act in-bundling PKC substrate MARCKS in confluent MDCK cells originating from t he distal tubulus of dog kidney, LLC-PK1 cells originating from the proxima l tubulus of pig kidney, src-transformed DCK cells, epidermoid carcinoma A4 31 cells, and MDCK cells grown in low calcium medium (LC medium) in low den sity were visualized with phase contrast and immunofluorescence microscopy. Four different responses to the PMA-treatment in actin-based structures of cultured epithelial cells were observed: 1) disintegration of the membrane skeleton in confluent MDCK cells; 2) depolymerization of the stress fibers in confluent MDCK and LLC-PK1 cells, 3) formation of the membrane skeleton in A431 cells, and 4) formation of the stress fibers and membrane skeleton in LC-MDCK cells. Thus, it seems that in fully confluent tightly sealed ep ithelium, activation of PKC has a deleterious effect on actin-based structu res, whereas in cells without contacts or loose contacts, activation of PKC by PMA results in improvement of actin-based cytoskeletal structures. The main difference between the two kidney cell lines used is their selectivity to ion transport: the monolayer of LLC-PK1 cells is anion selective and MD CK cells cation selective. We propose a model where alterations in the ioni c milieu within the MDCK cells by means of cation channels affect the disin tegration of the membrane skeleton. The distribution of MARCKS followed the distribution of fodrin in both cell lines upon PMA-treatment, suggesting t hat phosphorylation of MARCKS by PKC may contribute in the regulation of th e integrity of the membrane skeleton. J. Cell. Physiol. 181:83-95, 1999. (C ) 1999 Wiley-Liss, Inc.