Nucleocytoplasmic Interactions and Dynamics in Emery-Dreifuss Muscular Dystrophy

描述(申请人提供)：Emery-Dreifuss肌营养不良症(EDMD)是由核膜蛋白编码基因突变引起的。常染色体EDMD是由编码A-型层蛋白的LMNA突变和编码Emerin的EMD突变导致的X连锁EDMD。Nesprint和Sun编码基因的突变也与EDMD表型有关。这些基因编码的蛋白质参与连接细胞核内部和细胞质，形成或结合LINC复合体。我们已经证明，导致常染色体EDMD的LMNA突变以及A-型层蛋白的缺乏，通过破坏细胞核与TAN系的锚定来阻止细胞质肌动蛋白依赖的核运动，TAN系由SUN2和Nesprin-2组成。发生在X连锁的EDMD中的Emerin的丢失，通过改变驱动它的细胞质肌动蛋白逆行流动，额外地阻止了核的运动。太阳和太阳的耗尽也通过改变TAN线功能而导致有缺陷的核运动。在缺乏A型lamins的细胞中，抑制ERK1/2可以逆转核运动缺陷。ERK1/2由lamins、Emerin、Suns和Nesprint以及常染色体EDMD动物模型的横纹肌中的变化激活。我们现在建议检验这一假设，即导致EDMD的基因改变与它们所产生的核运动缺陷和ERK1/2激活之间存在联系。为了验证这一假设，我们将利用一种创新的基于成纤维细胞的分析方法来机械地解剖核运动中的缺陷，然后将发现扩展到体内培养的成肌细胞和骨骼肌。在目标1中，我们将研究LINC复合体功能、ERK1/2活性和核运动之间的关系，重点是过度激活的ERK1/2如何阻止核运动。在目标2中，我们将研究在生理激活过程中细胞核运动与ERK1/2活性之间的时间关系，并进行一系列生物物理实验，以直接测试移动细胞核是否调节ERK1/2信号。在目标3和4中，我们将把我们的研究扩展到肌肉细胞，检查LINC复合蛋白和ERK1/2如何影响移行性成肌细胞的核运动，并确定A型Lamins和Emerin是否影响肌肉中正确的核定位，以及在Lamins或Emerin变化的EDMD小鼠模型中。总体而言，这项研究将为EDMD的细胞病理学提供新的见解，同时发现有关核运动的新信息，这是一种对基础细胞生物学具有广泛意义的细胞功能。

DESCRIPTION (provided by applicant): Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in genes encoding proteins of the nuclear envelope. Autosomal EDMD results from mutations in LMNA, which encodes A-type lamins, and X-linked EDMD from mutations in EMD, which encodes emerin. Mutations in genes encoding nesprins and SUNs have also been associated with the EDMD phenotype. The proteins encoded by these genes are involved in connecting the inside of the nucleus to the cytoplasm, forming or associating with the LINC complex. We have shown that LMNA mutations causing autosomal EDMD, as well as deficiency of A-type lamins, block cytoplasmic actin-dependent nuclear movement by disrupting anchoring of the nucleus to structures known as TAN lines, which are composed of SUN2 and nesprin-2. Loss of emerin, as occurs in X-linked EDMD, additionally blocks nuclear movement by altering retrograde cytoplasmic actin flow that drives it. Depletion of SUNs and nesprins also leads to defective nuclear movement by altering TAN line function. In cells lacking A- type lamins, inhibition of ERK1/2, which is activated by alterations in lamins, emerin, SUNs and nesprins as well as in striated muscle of animal models of autosomal EDMD, reverses the nuclear movement defect. We now propose to test the hypothesis that there is a link between genetic alterations that cause EDMD and the nuclear movement defects and ERK1/2 activation they create. To test this hypothesis, we will utilize an innovative fibroblast-based assay to mechanistically dissect defects in nuclear movement and then extend the findings to cultured myoblasts and skeletal muscle in vivo. In Aim 1, we will examine the relationship between LINC complex function, ERK1/2 activity and nuclear movement, with a focus on how hyperactivated ERK1/2 prevents nuclear movement. In Aim 2, we will examine the temporal relationship between nuclear movement and ERK1/2 activity during physiological activation of the kinase and perform a series of biophysical experiments to directly test if moving the nucleus regulates ERK1/2 signaling. In Aims 3 and 4, we will extend our studies into muscle cells, examining how LINC complex proteins and ERK1/2 affect nuclear movement in migrating myoblasts and determining if A-type lamins and emerin affect proper nuclear positioning in muscle, in mouse models of EDMD with alterations in lamins or emerin. Overall, this research will provide novel insights into the cellular pathology of EDMD and simultaneously uncover new information about nuclear movement, a cellular function of broad significance to basic cell biology.