Polarisation-dependent association of plectin with desmoplakin and the lateral submembrane skeleton in MDCK cells.

The intermediate filament-binding protein plectin and cytokeratin were localised at the cellular periphery of fully polarised Madin-Darby canine kidney (MDCK) cells, whereas vimentin was primarily found in a perinuclear network. Confocal and immunoelectron microscopy revealed that plectin was restricted to areas underlying the lateral plasma membrane. It colocalised with fodrin, a component of the submembrane skeleton, and was closely associated with desmosomal plaque structures. Biochemically, plectin was shown to interact directly with immunoprecipitated desmoplakin in vitro. Upon loss of cell polarity in low calcium medium, plectin redistributed to a cytoplasmic vimentin- and cytokeratin-related network, clearly distinct from diffusely distributed fodrin and internalised desmoplakin structures. The structural reorganisation of plectin was also reflected by an increased solubility of the protein in Triton X-100/high salt, and a decrease in its half-life from approximately 20 to approximately 5 hours. Furthermore, unlike cytokeratins and vimentin, desmoplakin and fodrin did not associate with plectin attached to magnetic beads in cell lysates of unpolarised cells, while all proteins formed a stable complex in polarised cells. Altogether, these data indicate that plectin is involved in the anchorage of intermediate filaments to desmosomes and to the submembrane skeleton in polarised MDCK cells.