Mutations in LMNA Modulate the Lamin A - Nesprin-2 Interaction and Cause LINC Complex Alterations

In eukaryotes the genetic material is enclosed by a continuous membrane system, the nuclear envelope (NE). Along the NE specific proteins assemble to form meshworks and mutations in these proteins have been described in a group of human diseases called laminopathies. Laminopathies include lipodystrophies, muscle and cardiac diseases as well as metabolic or progeroid syndromes. Most laminopathies are caused by mutations in the LMNAgene encoding lamins A/C. Together with Nesprins (Nuclear Envelope Spectrin Repeat Proteins) they are core components of the LINC complex (Linker of Nucleoskeleton and Cytoskeleton). The LINC complex connects the nucleoskeleton and the cytoskeleton and plays a role in the transfer of mechanically induced signals along the NE into the nucleus, and its components have been attributed functions in maintaining nuclear and cellular organization as well as signal transduction.; Here we narrowed down the interaction sites between lamin A and Nesprin-2 to aa 403-425 in lamin A and aa 6146-6347 in Nesprin-2. Laminopathic mutations in and around the involved region of lamin A (R401C, G411D, G413C, V415I, R419C, L421P, R427G, Q432X) modulate the interaction with Nesprin-2 and this may contribute to the disease phenotype. The most notable mutation is the lamin A mutation Q432X that alters LINC complex protein assemblies and causes chromosomal and transcription factor rearrangements.; Mutations in Nesprin-2 and lamin A are characterised by complex genotype phenotype relations. Our data show that each mutation in LMNAanalysed here has a distinct impact on the interaction among both proteins that substantially explains how distinct mutations in widely expressed genes lead to the formation of phenotypically different diseases

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 Ca2+/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-B. We show that their nuclear transport is unlikely due to the canonical RAN mediated nuclear import, but rather to a RAN independent Ca2+/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 Ca2+ concentrations in the cytoplasm suggesting a previously unidentified role of Nesprins in Ca2+ 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 Ca2+. Taken together, the data suggests a novel role of Nesprin-2 in Ca2+/Calmodulin mediated nuclear trafficking and provides new insights which can guide future therapies

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

Schematic of lamin A mutations.

<p>(<b>A</b>) aa residues 399 to 434 of WT lamin A are shown. The interaction site of lamin A to Nesprin-2 is given in green. Below, the WT sequence mutations analysed in the present study are highlighted. Similarities to the WT sequence are shown as black lines, exchanged aa are indicated in red. The mutation Q432X leads to a stop codon (X). (<b>B</b>) Western blot analysis of GFP lamin A fusion proteins. The proteins were transiently expressed in COS7 cells, lysates were analysed by SDS-PAGE followed by western blot with mAb K3-184 that confirmed the predicted molecular weights (given in kDa on the left). <i>WB</i> – western blot.</p

The perinuclear actin cap is not altered in the presence of GFP lamin A Q432X or V415I.

<p>C2C12 cells transiently expressing WT GFP lamin A, the mutant proteins Q432X or V415I were stained with DAPI and TRITC phalloidin. The stainings were documented by using a confocal microscope and pictures were taken at the apical plane (red), the mid plane (yelow) and the basal plane (gray). Actin cap fibers are present in control or mutant lamin A expressing cells (long arrow). TRITC phalloidin was present in some GFP lamin A Q432X aggregates (short arrow).</p

The interaction site of lamin A to Nesprin-2 is in a loop and mutations in <i>LMNA</i> modulate the interaction to Nesprin-2.

<p>(<b>A</b>) Amino acid alignment of a human lamin A/C amino acids 351–490 (HsLaminA; uniprot accession number P02545) and the corresponding sequence in human lamin B (HsLamin; P20700) and <i>Mus musculus</i> lamin A (MmLaminA; P48678). Secondary structure elements are shown on top of the alignment and the lamin A region that interacts with Nesprin-2 is boxed in green. Conserved residues are highlighted black, similar residues are boxed. (<b>B</b>) Prediction of the three dimensional structure of human lamin A aa 351–490. The three dimensional structure of human Lamin A^351–490 was predicted by using multiple PDB structures as templates (1UFGA, 1IFRA, and 2LLA) in the MULTICOM server <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071850#pone.0071850-Wang1" target="_blank">[41]</a>. The lamin A interaction site aa 403–425 to Nesprin-2 is highlighted in green and aa 403 and 425 are pointed out in blue. Amino acids that targets for the mutations analysed here are highlighted in red. The structure prediction was generated by using pyMOL v1.3. (<b>C</b>) The binding properties between WT GST Nesprin-2 SR52,53 and GFP lamin A mutations were analysed by pull down experiments. COS7 cells expressing WT or mutated GFP lamin A proteins were lysed and incubated with recombinant GST Nesprin-2 SR52,53 proteins. GST Nesprin-2 SR55,56 proteins were used as negative controls. (<b>D</b>) WT and mutant GFP lamin A proteins show distinct binding properties to WT GST Nesprin-2 SR52,53. The zero baseline represents the 100% binding affinity between WT GST Nesprin-2 SR52,53 and GFP lamin A (for details see materials and methods). Deviations caused by distinct mutations in <i>LMNA</i> are given in percent. Each mutation was analysed by four to seven independent experiments.</p

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

Lamin A/C dependent localization of the largest nesprin-2 isoform NUANCE, a giant scaffolder at the nuclear envelope

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