Nesprin-2 giant safeguards nuclear envelope architecture in LMNA S143F progeria cells

Abstract

The S143F lamin A/C point mutation causes a phenotype combining features of myopathy and progeria. We demonstrate here that patient dermal fibroblast cells have dysmorphic nuclei containing numerous blebs and lobulations, which progressively accumulate as cells age in culture. The lamin A/C organization is altered, showing intranuclear and nuclear envelope (NE) aggregates and presenting often a honeycomb appearance. Immunofluorescence microscopy showed that nesprin-2 C-terminal isoforms and LAP2alpha were recovered in the cytoplasm, whereas LAP2beta and emerin were unevenly localized along the NE. In addition, the intranuclear organization of acetylated histones, histone H1 and the active form of RNA polymerase II were markedly different in patient cells. A subpopulation of mutant cells, however, expressing the 800 kDa nesprin-2 giant isoform, did not show an overt nuclear phenotype. Ectopic expression of p.S143F lamin A in fibroblasts recapitulates the patient cell phenotype, whereas no effects were observed in p.S143F LMNA keratinocytes, which highly express nesprin-2 giant. Overexpression of the mutant lamin A protein had a more severe impact on the NE of nesprin-2 giant deficient fibroblasts when compared with wild-type. In summary, our results suggest that the p.S143F lamin A mutation affects NE architecture and composition, chromatin organization, gene expression and transcription. Furthermore, our findings implicate a direct involvement of the nesprins in laminopathies and propose nesprin-2 giant as a structural reinforcer at the NE.

Gene-rich chromosomal regions are preferentially localized in the lamin B deficient nuclear blebs of atypical progeria cells

“…This particular mutation was selected as patient's cells typically contain a single large nuclear bleb even in early passage cultures. 19 Nuclear blebs in p.S143F cells were devoid of B-type lamins and similar to blebs seen in progerin expressing cells. 9 The large size of these p.S143F-associated LA enriched bleb www.taylorandfrancis.comdomains represents an ideal model for studies of their structural and functional properties.…”

“…This missense mutation is in close proximity to the E145K mutation but results in a progeroid and myopathy phenotype and the cells typically contain nuclei with one large bleb. 18,19 Our results show that gene-rich chromosomal regions are preferentially located in blebs and that transcription is not globally inhibited or reduced in this compartment. However, we have observed a loss of the co-activator SKIP, from the lamina region of nuclear blebs when compared to the remainder of the nucleus in p.S143F fibroblasts.…”

“…1A). 18,19 The number of cells with nuclear blebs increased with the accumulation of passage number and was »22% by passage number 15 (p15) and »60% by p40. 69% of cells with abnormally shaped nuclei showed a single large nuclear bleb, while 19% showed 2 blebs and 12% 3 or more blebs (n > 200).…”

“…36 For example, it has been reported that nesprin-2 is greatly reduced throughout the nuclear periphery including the blebs and that the re-expression of the nesprin-2 giant isoform restores a normal nuclear phenotype in p.S143F progeria cells. 19 For future research, it will be very important to study the impact of the LINC complex on nuclear shape changes and their role in other laminopathies. 36 Our results also show that the LA/C-rich blebs in p.S143F nuclei are devoid of centromeric heterochromatin and are enriched in euchromatin.…”

“…For this purpose, we chose a probe that spans a transcriptionally active region on chromosome 19. RNA FISH signals were detected throughout the nucleoplasm including the bleb region, and larger foci likely representing the actual transcription loci were occasionally detected (Fig.…”