Nesprin-2 Giant (NUANCE) maintains nuclear envelope architecture and composition in skin.

Giant isoforms, encoded by Nesprin-1 (Syne1) and Nesprin-2 (Syne2), are multifunctional actin-binding and nuclear-envelope-associated proteins belonging to the spectrin superfamily. Here, we investigate the function of Nesprin-2 Giant (NUANCE) in skin by generating mice lacking the actin-binding domain of Nesprin-2 (Nesprin-2DeltaABD). This loss results in a slight but significant thickening of the epidermis, which is a consequence of the increased epithelial nuclear size. Nonetheless, epidermal proliferation and differentiation appear normal in the knockout epidermis. Surprisingly, Nesprin-2 C-terminal-isoform expression and nuclear envelope localization were affected in certain tissues. Nuclei of primary dermal knockout fibroblasts and keratinocytes were heavily misshapen, displaying a striking similarity to nuclear deformations characteristic of laminopathies. Furthermore, emerin, the protein involved in the X-linked form of Emery-Dreifuss muscular dystrophy (EDMD), was unevenly distributed along the nuclear envelope in mutant fibroblasts, often forming aggregates in the deformed nuclear envelope areas. Thus, Nesprin-2 is an important scaffold protein implicated in the maintenance of nuclear envelope architecture. Aged knockout fibroblasts readily generated, by alternative splicing and alternative translation initiation, aberrant Nesprin-2 Giant isoforms that lacked an ABD but that were sufficient to restore nuclear shape and emerin localization; this suggests that other regions of Nesprin-2 Giant, potentially including its spectrin repeats, are crucial for these functions.

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

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.