Selective accumulation of IF proteins at a focal juxtanuclear site in COS-1 cells transfected with mouse keratin 18 cDNA

Abstract

COS-1 cells contain two keratins analogous to human keratins 8 (type II) and 18 (type I), and vimentin. Transfection of a plasmid, pSVK18, containing a mouse keratin 18 cDNA regulated by the SV40 early region promoter, was used to force the synthesis of exogenous (but homologous) type I keratin and to assess the effect of the oversynthesis of a keratin on endogenous keratins and vimentin intermediate filaments (IFs). Double immunofluorescence microscopy with monoclonal antimouse keratin 18 and monoclonal anti-human keratins 8 and 18 antibodies which cross-react with monkey keratins, showed that mouse keratin 18 formed typical IFs with the endogenous keratins but also accumulated in a focal area near the nucleus. Vimentin and its associated protein, p50, also colocalized at the juxtanuclear focal region, but the vimentin IFs of the outer cytoplasm vanished. Similar analyses with anti-tubulin and anti-actin antibodies indicated that the accumulated mouse keratin 18 colocalized with the centrosome but did not disturb the organization of microtubules or microfilaments. Anti-lamin and anti-SV40 large T antibodies showed that the oversynthesis of mouse keratin 18 had no effect on the distribution of these proteins. The accumulation was therefore selective for the cytoplasmic IF proteins. Electron microscopy and immunogold labeling of whole-mount detergent-extracted cells demonstrated that the accumulated IFs in the centrosomal region extended as a dense IF plexus-like network anchored to part of the nuclear surface.