The role of nucleo-cytoskeletal link proteins in skeletal muscle

核细胞骨架连接蛋白在骨骼肌中的作用

• 批准号: 8693926
• 负责人: Ju Chen
• 金额: $ 32.7万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8693926
• 关键词: Ablation; Address; Adult; Age-Months; Binding; Biochemical; Biological Process; Biology; Body Weight; C-terminal; Cell Nucleus; Cells; Congenital Heart Defects; Contracture; Cytoskeleton; Data; Defect; Desmin; Emery-Dreifuss Muscular Dystrophy; Exercise; Exhibits; Family; Gene Expression; Genes; Genetically Engineered Mouse; Goals; In Vitro; Intermediate Filaments; Kinetics; Knock-out; Knockout Mice; Lamin Type A; Light; Link; LoxP-flanked allele; Mechanics; Membrane Proteins; Microfilaments; Modeling; Molecular; Mus; Muscle; Muscle Fibers; Muscle Weakness; Muscle function; Muscular Dystrophies; Mutant Strains Mice; Mutation; Myoblasts; Myocardium; Myopathy; N-terminal; Nuclear; Nuclear Envelope; Nuclear Inner Membrane; Nuclear Lamina; Nuclear Outer Membrane; Null Lymphocytes; Organism; Pathogenesis; Patients; Perinatal; Phenotype; Physiological; Positioning Attribute; Protamines; Protein Family; Proteins; Respiratory Failure; Role; Signal Transduction; Skeletal Muscle; Skeletal Myoblasts; Spectrin; Stretching; Survivors; Synapses; Tamoxifen; Testing; calponin; emerin; improved; in vivo; insight; link protein; loss of function; member; mutant; research study; retinal rods; satellite cell

DESCRIPTION (provided by applicant): Recent data suggest that mutations in Nesprin 1 and 2 may be involved in the pathogenesis of Emery-Dreifuss muscular dystrophy (EDMD), which is characterized by progressive skeletal muscle weakness with associated muscle contractures, and variable cardiac defects. 40% of patients with EDMD have been shown to have mutations in Emerin or Lamin A/C, two genes encoding proteins localized to the inner nuclear membrane (INM) and its underlying lamina, respectively. The INM, the outer nuclear membrane (ONM), and the nuclear lamina comprise the nuclear envelope, which is linked to the cytokeleton by members of both the SUN and Nesprin protein families. Approximately 60% of EDMD patients do not have mutations in either Emerin or LMNA. Intriguingly, mutations in Nesprin 1 and 2 have been associated with EDMD. Nesprins belong to a newly discovered family of mammalian spectrin-repeat proteins. Mice lacking Lamin A/C exhibit features of EDMD. Studies on skeletal muscle cultures isolated from these mice have demonstrated a critical role for Lamin A/C in skeletal myoblast differentiation and in mechanical stiffness by maintaining nucleo-cytoskeletal integrity. In contrast, Emerin knockout mice do not exhibit EDMD. However, in skeletal muscle cultures from Emerin null mice, myoblast differentiation is perturbed to the same extent as observed in Lamin A/C null cells. Moreover, although Emerin null cells do not exhibit decreased mechanical stiffness as found in Lamin A/C null cells, perturbations in gene expression associated with mechanotransduction are observed. Both Nesprin 1 and 2 are ubiquitously expressed. To investigate the functional roles of Nesprin 1 and 2, we have generated floxed alleles for Nesprin 1 and 2. By utilizing protamine Cre mice, we have generated global loss of function mutants for Nesprin 1 and 2 (Nesprin 1-/- and Nesprin 2-/-). Our studies reveal that Nesprin 2-/- mice are viable and have no obvious basal phenotype, whereas approximately 60% of Nesprin 1-/- mice die perinatally. Remaining survivors have reduced body weight and compromised exercise capacity. We also found 100% perinatal lethality in Nesprin 1-/-;Nesprin 2-/- double mutant mice. Histological analyses of Nesprin 1-/- mice and Nesprin 1-/-;Nesprin 2-/- mice revealed abnormal positioning of non-synaptic nuclei and disappearance of clusters of synaptic nuclei. The overall goals of this proposal are to test the hypothesis that Nesprin 1 and 2 have distinct and overlapping roles in skeletal muscle nuclear positioning, nuclear membrane integrity, skeletal myoblast differentiation, mechanical stiffness, mechano-transduction, and muscle function. We will achieve these goals by comprehensive molecular, biochemical, histological, and physiological analysis of the skeletal muscle phenotypes in our four existing genetically engineered mouse lines. Results will shed light into mechanisms by which mutations in Nesprin contribute to myopathies.

描述（由申请人提供）：最近的数据表明，Nesprin 1和2中的突变可能参与Emery-Dreifuss肌营养不良症（EDMD）的发病机制，其特征在于进行性骨骼肌无力伴相关肌肉挛缩和可变心脏缺陷。40%的EDMD患者已被证明在Emerin或Lamin A/C中存在突变，这两种基因分别编码位于内核膜（INM）及其下层的蛋白质。INM、外核膜（ONM）和核纤层构成核膜，核膜通过SUN和Nesprin蛋白家族的成员与细胞骨架连接。大约60%的EDMD患者在Emerin或LMNA中没有突变。有趣的是，Nesprin 1和2的突变与EDMD相关。Nesprins属于一个新发现的哺乳动物血影蛋白重复蛋白家族。缺乏核纤层蛋白A/C的小鼠表现出EDMD的特征。对从这些小鼠分离的骨骼肌培养物的研究已经证明了核纤层蛋白A/C在骨骼肌成肌细胞分化和通过维持核细胞骨架完整性而在机械刚度中的关键作用。相比之下，Emerin敲除小鼠不表现出EDMD。然而，在来自Emerin无效小鼠的骨骼肌培养物中，成肌细胞分化受到与在核纤层蛋白A/C无效细胞中观察到的相同程度的干扰。此外，尽管裸细胞不表现出如核纤层蛋白A/C裸细胞中发现的降低的机械刚度，但观察到与机械转导相关的基因表达的扰动。Nesprin 1和2均广泛表达。为了研究Nesprin 1和2的功能作用，我们已经产生了Nesprin 1和2的floxed等位基因。通过利用鱼精蛋白Cre小鼠，我们已经产生了Nesprin 1和2的功能缺失突变体（Nesprin 1-/-和Nesprin 2-/-）。我们的研究表明，Nesprin 2-/-小鼠是可行的，没有明显的基础表型，而大约60%的Nesprin 1-/-小鼠围产期死亡。其余幸存者体重减轻，运动能力受损。我们还发现Nesprin 1-/-;Nesprin 2-/-双突变小鼠的围产期致死率为100%。Nesprin 1-/-小鼠和Nesprin 1-/-;Nesprin 2-/-小鼠的组织学分析显示非突触核的异常定位和突触核簇的消失。本提案的总体目标是检验Nesprin 1和2在骨骼肌核定位、核膜完整性、骨骼肌成肌细胞分化、机械刚度、机械转导和肌肉功能中具有不同和重叠作用的假设。我们将通过对现有的四种基因工程小鼠品系的骨骼肌表型进行全面的分子、生物化学、组织学和生理学分析来实现这些目标。结果将揭示Nesprin突变导致肌病的机制。

项目成果

Obscurin is required for ankyrinB-dependent dystrophin localization and sarcolemma integrity.

依赖Ankyrinb的肌营养不良蛋白定位和肌膜完整性所必需。

• DOI: 10.1083/jcb.201205118
• 发表时间: 2013-02-18
• 期刊: The Journal of cell biology
• 作者: Randazzo D;Giacomello E;Lorenzini S;Rossi D;Pierantozzi E;Blaauw B;Reggiani C;Lange S;Peter AK;Chen J;Sorrentino V
• 通讯作者: Sorrentino V

Ankrd2 is a modulator of NF-κB-mediated inflammatory responses during muscle differentiation.

• DOI: 10.1038/cddis.2013.525
• 发表时间: 2014-01-16
• 期刊: Cell death & disease
• 影响因子: 9