De novo formation of cytokeratin filaments in calf lens cells and cytoplasts after transfection with cDNAs or microinjection with mRNAs encoding human cytokeratins.

We have studied the formation of new intermediate-sized filaments (IFs) by human cytokeratins (CKs) 8 and/or 19 in cultured bovine lens cells stably transfected with the corresponding cDNAs under SV40 promoter control. In the transfected cells, polypeptides of both type I and type II CKs were synthesized to near-equimolar amounts, formed heterotypic complexes and assembled into IFs with a peculiar tendency to accumulate into variously sized, often roundish aggregates in the juxtanuclear region, usually one per cell. Electron microscopy of these large CK IF aggregates revealed typical 7 to 12-nm IFs, tightly packed together in an apparently haphazard mode. By immunoelectron microscopy, the CK IFs could be readily distinguished from the vimentin IFs which were abundant in these cells. Electron microscopy also showed that many of the CK IF aggregates were located in the vicinity of the nucleus but did not have direct contact with the nuclear envelope; moreover, their location did not regularly correspond to those of the centrosomes and the Golgi apparatus. During enucleation of transfected cells in the presence of cytochalasin B, the CK aggregates were often retained in the cytoplast. After microinjection of CK 8 and 19 mRNAs, synthesized in vitro from cDNA molecules, into enucleated cytoplasts prepared from untransfected cells, CK IFs similar to those observed in microinjected whole cells were formed but often showed a wider cytoplasmic distribution. Our observations indicate that typical CK IFs can form, in vivo, in the absence of any nuclear structures. We discuss possible reasons for the tendency of the CK IFs to accumulate, in this cell line, into a juxtanuclear aggregate, in relation to similar CK-IF aggregates formed in certain normal cell types and upon toxic damages.