The roles of the different molecular domains of intermediate filament (IF) proteins in the assembly and higher order organization of IF structures have recently been studied by various groups but with partially controversial results. To examine the requirement of the aminoterminal (head) and the carboxyterminal (tail) domain of cytokeratins (CKs) for de novo IF formation in the living cell, we have constructed cDNAs coding for intact as well as head- and/or tail-less human CKs 8 and 18 and the naturally tail-less human CK 19, all under the control of the human beta-actin promoter. After transient and stable transfections of mouse 3T3-L1 cells, which are devoid of any CKs, we have studied, with such constructs, the resulting gene products by gel electrophoresis and immunolocalization techniques. By light and electron microscopy we show that extended cytoplasmic IF meshworks are formed from pairs of the type II CK 8 with the type I CKs 18 or 19 as well as from pairs of tail-less CK 8 with tail-less CKs 18 or 19 in the transfected cells, proving that the absence of the tail domain in both types of CKs does not prevent the de novo formation of regular IFs. Most surprisingly, however, we have observed spectacular alterations in the nucleocytoplasmic distribution of the IFs formed from tail-less CKs. In many of the transfected cells, a large part, or all, of the detectable CKs was found to occur in extensive IF bundles in the nucleoplasm. Intranuclear accumulations of CK deposits, however mostly nonfibrillar, were also observed when the cells had been transfected with cDNAs encoding tail-less CKs also lacking their head domains, whereas CKs deleted only in the head domain were found exclusively in the cytoplasm. The specific domain requirements for the assembly of cytoplasmic IF bundles are discussed and possible mechanisms of intranuclear accumulation of IFs are proposed.
Engineering Improved Photoswitches for the Control of Nucleocytoplasmic Distribution