RESUMEN
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.
Asunto(s)
Núcleo Celular/metabolismo , Epidermis/metabolismo , Células Epiteliales/metabolismo , Proteínas de Microfilamentos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Membrana Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Empalme Alternativo/genética , Animales , Animales Recién Nacidos , Diferenciación Celular/fisiología , Núcleo Celular/ultraestructura , Polaridad Celular/genética , Forma de la Célula/genética , Células Cultivadas , Expansión de las Repeticiones de ADN/genética , Epidermis/anomalías , Epidermis/ultraestructura , Células Epiteliales/ultraestructura , Fibroblastos/metabolismo , Fibroblastos/ultraestructura , Humanos , Queratinocitos/metabolismo , Queratinocitos/ultraestructura , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Proteínas de Microfilamentos/química , Proteínas de Microfilamentos/genética , Mutación/genética , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Membrana Nuclear/ultraestructura , Proteínas Nucleares/química , Proteínas Nucleares/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estructura Terciaria de Proteína/genéticaRESUMEN
Nesprins form a novel class of nuclear envelope-anchored spectrin-repeat proteins. We show that a direct association of their highly conserved C-terminal luminal domain with the inner nuclear membrane protein Sun1 mediates their nuclear envelope localisation. In Nesprin-1 and Nesprin-2 the conserved C-terminal amino acids PPPX are essential for the interaction with a C-terminal region in Sun1. In fact, Sun1 is required for the proper nuclear envelope localisation of Nesprin-2 as shown using dominant-negative mutants and by knockdown of Sun1 expression. Sun1 itself does not require functional A-type lamins for its localisation at the inner nuclear membrane in mammalian cells. Our findings propose a conserved nuclear anchorage mechanism between Caenorhabditis elegans and mammals and suggest a model in which Sun1 serves as a ;structural bridge' connecting the nuclear interior with the actin cytoskeleton.
Asunto(s)
Proteínas de Microfilamentos/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Membrana Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Animales , Células COS , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Núcleo Celular/metabolismo , Chlorocebus aethiops , Clonación Molecular , Proteínas del Citoesqueleto , Células HeLa , Humanos , Membranas Intracelulares/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C3H , Ratones Noqueados , Proteínas de Microfilamentos/genética , Proteínas Asociadas a Microtúbulos/genética , Modelos Biológicos , Proteínas del Tejido Nervioso/genética , Membrana Nuclear/clasificación , Membrana Nuclear/ultraestructura , Proteínas Nucleares/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
The vertebrate proteins Nesprin-1 and Nesprin-2 (also referred to as Enaptin and NUANCE) together with ANC-1 of Caenorhabditis elegans and MSP-300 of Drosophila melanogaster belong to a novel family of alpha-actinin type actin-binding proteins residing at the nuclear membrane. Using biochemical techniques, we demonstrate that Nesprin-2 binds directly to emerin and the C-terminal common region of lamin A/C. Selective disruption of the lamin A/C network in COS7 cells, using a dominant negative lamin B mutant, resulted in the redistribution of Nesprin-2. Furthermore, using lamin A/C knockout fibroblasts we show that lamin A/C is necessary for the nuclear envelope localization of Nesprin-2. In normal skin where lamin A/C is differentially expressed, strong Nesprin-2 expression was found in all epidermal layers, including the basal layer where only lamin C is present. This indicates that lamin C is sufficient for proper Nesprin-2 localization at the nuclear envelope. Expression of dominant negative Nesprin-2 constructs and knockdown studies in COS7 cells revealed that the presence of Nesprin-2 at the nuclear envelope is necessary for the proper localization of emerin. Our data imply a scaffolding function of Nesprin-2 at the nuclear membrane and suggest a potential involvement of this multi-isomeric protein in human disease.
