The Nesprin-1/-2 ortholog ANC-1 regulates organelle positioning in C. elegans independently from its KASH or actin-binding domains.
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ABSTRACT:
SUBMITTER: Hao H
PROVIDER: S-EPMC8139857 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Nesprin 1 is an outer nuclear membrane protein that is thought to link the nucleus to the actin cytoskeleton. Recent data suggest that mutations in Nesprin 1 may also be involved in the pathogenesis of Emery-Dreifuss muscular dystrophy. To investigate the function of Nesprin 1 in vivo, we generated a mouse model in which all isoforms of Nesprin 1 containing the C-terminal spectrin-repeat region with or without KASH domain were ablated. Nesprin 1 knockout mice are marked by decreased survival rates, growth retardation and increased variability in body weight. Additionally, nuclear positioning and anchorage are dysfunctional in skeletal muscle from knockout mice. Physiological testing demonstrated no significant reduction in stress production in Nesprin 1-deficient skeletal muscle in either neonatal or adult mice, but a significantly lower exercise capacity in knockout mice. Nuclear deformation testing revealed ineffective strain transmission to nuclei in muscle fibers lacking Nesprin 1. Overall, our data show that Nesprin 1 is essential for normal positioning and anchorage of nuclei in skeletal muscle.
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Project description:Many nuclear positioning events involve linker of nucleoskeleton and cytoskeleton (LINC) complexes, which transmit forces generated by the cytoskeleton across the nuclear envelope. LINC complexes are formed by trans-luminal interactions between inner nuclear membrane SUN proteins and outer nuclear membrane KASH proteins, but how these interactions are regulated is poorly understood. We combine in vivo C. elegans genetics, in vitro wounded fibroblast polarization, and in silico molecular dynamics simulations to elucidate mechanisms of LINC complexes. The extension of the KASH domain by a single alanine residue or the mutation of the conserved tyrosine at -7 completely blocked the nuclear migration function of C. elegans UNC-83. Analogous mutations at -7 of mouse nesprin-2 disrupted rearward nuclear movements in NIH 3T3 cells, but did not disrupt ANC-1 in nuclear anchorage. Furthermore, conserved cysteines predicted to form a disulfide bond between SUN and KASH proteins are important for the function of certain LINC complexes, and might promote a developmental switch between nuclear migration and nuclear anchorage. Mutations of conserved cysteines in SUN or KASH disrupted ANC-1-dependent nuclear anchorage in C. elegans and Nesprin-2G-dependent nuclear movements in polarizing fibroblasts. However, the SUN cysteine mutation did not disrupt nuclear migration. Moreover, molecular dynamics simulations showed that a disulfide bond is necessary for the maximal transmission of cytoskeleton-generated forces by LINC complexes in silico. Thus, we have demonstrated functions for SUN-KASH binding interfaces, including a predicted intermolecular disulfide bond, as mechanistic determinants of nuclear positioning that may represent targets for regulation.
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Project description:BackgroundNesprin-1-giant (1008kD) is a protein of the outer nuclear membrane that links nuclei to the actin cytoskeleton via amino-terminal calponin homology domains. The short nesprin-1 isoform, nesprin-1-α2, is present only in skeletal and cardiac muscle and several pathogenic mutations occur within it, but the functions of this short isoform without calponin homology domains are unclear. The aim of this study was to determine mRNA levels and protein localization of nesprin-1-α2 at different stages of muscle development in order to shed light on its functions.ResultsmRNA levels of all known nesprin-1 isoforms with a KASH domain were determined by quantitative PCR. The mRNA for the 111 kD muscle-specific short isoform, nesprin-1-α2, was not detected in pre-differentiation human myoblasts but was present at significant levels in multinucleate myotubes. We developed a monoclonal antibody against the unique amino-terminal sequence of nesprin-1-α2, enabling specific immunolocalization for the first time. Nesprin-1-α2 protein was undetectable in pre-differentiation myoblasts but appeared at the nuclear rim in post-mitotic, multinucleate myotubes and reached its highest levels in fetal muscle. In muscle from a Duchenne muscular dystrophy biopsy, nesprin-1-α2 protein was detected mainly in regenerating fibres expressing neonatal myosin. Nesprin-1-giant was present at all developmental stages, but was also highest in fetal and regenerating fibres. In fetal muscle, both isoforms were present in the cytoplasm, as well as at the nuclear rim. A pathogenic early stop codon (E7854X) in nesprin-1 caused reduced mRNA levels and loss of protein levels of both nesprin-1-giant and (unexpectedly) nesprin-1-α2, but did not affect myogenesis in vitro.ConclusionsNesprin-1-α2 mRNA and protein expression is switched on during myogenesis, alongside other known markers of muscle differentiation. The results show that nesprin-1-α2 is dynamically controlled and may be involved in some process occurring during early myofibre formation, such as re-positioning of nuclei.
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