Influence of the expression of mutated B-type lamins on nuclear architecture and function

Abstract

The work presented here demonstrates new insights into the assembly mechanisms of the nuclear lamina. By generation and expression of several lamin mutants in mammalian cells it was possible to analyze the influence of distinct lamin domains on cellular localization and their assembly properties. Both partial and complete head deleted lamin B2 localized to the nuclear rim but highly impaired nuclear shape indicating that the head domain is dispensable for nuclear envelope localization and however, important for efficient lamin assembly. In contrast, tail deleted lamin B2 mutants did not incorporate into the nuclear rim and were distributed throughout the cytoplasm and the nucleoplasm. This suggests that the tail domain contains those elements that are necessary for effectively guiding these lamins to the nuclear envelope. However, tailless mutants did not impair the formation of a nuclear lamina. An exhaustive mutation trial of the individual mitotic phosphoacceptor sites flanking the central rod domain from serine to aspartic acid was performed in order to test if this would still allow the integration of these mutants into the nuclear lamina and if this would lead to the disassembly of the nuclear lamina. Notably, the mutant proteins were not incorporated into the lamina at all but instead they formed intranuclear aggregates when expressed in U2OS cells. Interestingly, the effect of nuclear aggregate formation was independent of both the position of the mutated site and the number of sites mutated. Live cell imaging experiments showed that the aggregates are rather dynamic structures that are able to fuse and occupy single large lamin territories. Co-transfection studies of “mitotic” lamin B1, “mitotic” lamin B2 and NLS-vimentin suggest that the aggregates are deposited in the interchromosomal domain compartment (ICD). However, intermingling of the proteins was not observed. Extraction experiments revealed that the aggregates were rather loosely connected to the nuclear matrix. Although the mechanism underlying aggregate formation of “mitotic” lamin B1, “mitotic” lamin B2, and NLS-vimentin remains elusive, our results strongly suggest the existence of nuclear “protein processing centers”. Their functions may relate to the prevention of macromolecular crowding as well as to the organization and distribution of nuclear proteins in general. Wild type and mutant lamins were also expressed in mouse embryonic stem (ES) cells. Their ability to differentiate into all specialized cell types found in the adult mouse and the exhibition and maintenance of a normal diploid complement of chromosomes make them a valuable tool for cell biological studies. As expected, both wild type lamin B1 and wild type lamin B2 localized to the nuclear rim. The stem cell status of the cells was not affected. Expression of lamin B2 deletion mutants in mouse ES cells showed similar effects as those observed in U2OS cells suggesting that lamin proteins are similarly processed and assembled into the nuclear lamina in both differentiated and ES cells. Additionally, a novel nonsense mutation in the lamin A gene (pR321X) cosegregating with dilated cardiomyopathy and cardiac rhythm disturbances was analyzed in both cultivated cells and cardiac tissue of affected patients. Neither nuclear abnormalities nor reduced expression of the wild type protein was observed. In line with a strong nonsense-mediated mRNA decay (NMD), i.e. the NMD-dependent reduction in the relative amount of mutant mRNA, the truncated protein was not found. The potential transient presence of this mutant protein could be uncovered, however, by inhibition of the proteasomal system. It is therefore suggested that NMD is not sufficient to completely prevent the expression of truncated lamin A and that even trace amounts of it may negatively interfere with structural and/or regulatory functions of lamin A/C eventually leading to the development of cardiomyopathy.