Depletion of Nesprin-2 is associated with an embryonic lethal phenotype in mice.

Nesprin-2 is a nuclear envelope component and provides a link between cytoskeletal components of the cytoplasm and the nucleoplasm. Several isoforms are generated from its gene Syne2. Loss of the largest isoform Nesprin-2 Giant in mice is associated with a skin phenotype and altered wound healing, loss of C-terminal isoforms in mice leads to cardiomyopathies and neurological defects. Here we attempted to establish mice with an inducible knockout of all Nesprin-2 isoforms by inserting shRNA encoding sequences targeting the N- and C-terminus into the ROSA26 locus of mice. This caused early embryonic death of the animals harboring the mutant allele, which was presumably due to leaky expression of the shRNAs. Mutant embryos were only observed before E13. They had an altered appearance and were smaller in size than their wild type littermates. From this we conclude that the Nesprin-2 gene function is crucial during embryonic growth, differentiation and organogenesis.

Functional Analysis of LINC Complexes in the Skin.

The genome in eukaryotic cells is encased by two intricate and interconnected concentric membranes, which together with the underlying nuclear lamina form the nuclear envelope (NE). Two fundamental macromolecular structures are embedded within the nuclear envelope: the nuclear pore (NPC) and the LINC complex. The former perforates the nucleus controlling biomolecule trafficking between the nucleoplasm and the cytoplasm, while the latter integrates the nucleus via the cytoskeleton to the extracellular matrix. LINC complex structural and functional integrity is of utmost importance for various fundamental cellular functions. Mechanical forces are relayed into the nuclear interior via the LINC complex, which controls lamina organization, chromosome dynamics, and genome organization and stability. Thus, LINC constituents play pivotal roles in cellular architecture including organelle positioning, cell movement, tissue assembly, organ homeostasis, and organismal aging. The LINC complex oligomeric core contains several multi-isomeric, multifunctional, and often tissue-specific proteins. Therefore, for a proper functional analysis, genetic mouse models are an invaluable resource. Herein, we focus on the LINC complex roles in the skin and describe methods that enable the successful isolation of primary embryonic fibroblast and newborn skin cells, which can be then investigated functionally in vitro.

Nesprin-2 Interacts with Condensin Component SMC2.

The nuclear envelope proteins, Nesprins, have been primarily studied during interphase where they function in maintaining nuclear shape, size, and positioning. We analyze here the function of Nesprin-2 in chromatin interactions in interphase and dividing cells. We characterize a region in the rod domain of Nesprin-2 that is predicted as SMC domain (aa 1436-1766). We show that this domain can interact with itself. It furthermore has the capacity to bind to SMC2 and SMC4, the core subunits of condensin. The interaction was observed during all phases of the cell cycle; it was particularly strong during S phase and persisted also during mitosis. Nesprin-2 knockdown did not affect condensin distribution; however we noticed significantly higher numbers of chromatin bridges in Nesprin-2 knockdown cells in anaphase. Thus, Nesprin-2 may have an impact on chromosomes which might be due to its interaction with condensins or to indirect mechanisms provided by its interactions at the nuclear envelope.

Nesprin-2 mediated nuclear trafficking and its clinical implications.

Nuclear translocation of proteins has a crucial role in the pathogenesis of cancer, Alzheimer disease and viral infections. A complete understanding of nuclear trafficking mechanisms is therefore necessary in order to establish effective intervention strategies. Here we elucidate the role of Nesprin-2 in Ca(2+)/Calmodulin mediated nuclear transport. Nesprin-2 is an actin-binding nuclear envelope (NE) protein with roles in maintaining nuclear structure and location, regulation of transcription and mechanotransduction. Upon depletion of Nesprin-2 using shRNA, HaCaT cells show abnormal localization of the shuttling proteins BRCA1 and NF-κB. We show that their nuclear transport is unlikely due to the canonical RAN mediated nuclear import, but rather to a RAN independent Ca(2+)/Calmodulin driven mechanism involving Nesprin-2. We report novel interactions between the actin-binding domain of Nesprin-2 and Calmodulin and between the NLS containing region of BRCA1 and Calmodulin. Strikingly, displacing Nesprins from the NE resulted in increased steady state Ca(2+) concentrations in the cytoplasm suggesting a previously unidentified role of Nesprins in Ca(2+) regulation. On comparing Nesprin-2 and BRCA1 localization in the ovarian cancer cell lines SKOV-3 and Caov-3, Nesprin-2 and BRCA1 were localized to the NE envelope and the nucleus in SKOV-3, respectively, and to the cytoplasm in Caov-3 cells. Fibroblasts obtained from EDMD5 (Emery Dreifuss muscular dystrophy) patients showed loss of Nesprin-2 from the nuclear envelope, corresponding reduced nuclear localization of BRCA1 and enhanced cytoplasmic Ca(2+). Taken together, the data suggests a novel role of Nesprin-2 in Ca(2+)/Calmodulin mediated nuclear trafficking and provides new insights which can guide future therapies.