Nucleocytoplasmic Interactions and Dynamics in Emery-Dreifuss Muscular Dystrophy

描述（由申请人提供）：Emery-Dreifuss肌营养不良症（EDMD）是由编码核膜蛋白的基因突变引起的。常染色体EDMD是由编码a型纤层蛋白的LMNA突变引起的，而x连锁EDMD是由编码emerin的EMD突变引起的。编码nesprins和sun的基因突变也与EDMD表型有关。这些基因编码的蛋白质参与连接细胞核内部与细胞质，形成或与LINC复合物结合。我们已经证明，导致常染色体EDMD的LMNA突变，以及a型纤层蛋白的缺乏，通过破坏细胞核锚定到由SUN2和nesprin2组成的TAN线结构，阻断细胞质肌动蛋白依赖的核运动。在x连锁EDMD中，emerin的缺失还会通过改变驱动核运动的逆行细胞质肌动蛋白流来阻止核运动。太阳和nesprins的耗竭也通过改变TAN线功能导致核运动缺陷。在缺乏A型纤层蛋白的细胞中，常染色体EDMD动物模型中ERK1/2（由纤层蛋白、emerin、SUNs和nesprins以及横纹肌的改变激活）的抑制逆转了核运动缺陷。我们现在提议测试导致EDMD的遗传改变与它们产生的核运动缺陷和ERK1/2激活之间存在联系的假设。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实验来机械解剖核运动缺陷，然后将研究结果扩展到培养的成肌细胞和体内骨骼肌。在Aim 1中，我们将研究LINC复合物功能、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Aim 2中，我们将研究在激酶生理激活过程中核运动与ERK1/2活性之间的时间关系，并进行一系列生物物理实验来直接测试核运动是否调节ERK1/2信号传导。在目标3和目标4中，我们将把我们的研究扩展到肌肉细胞，研究LINC复合物蛋白和ERK1/2如何影响迁移的成肌细胞的核运动，并确定a型层粘胶蛋白和emerin是否会影响层粘胶蛋白或emerin改变的EDMD小鼠模型中肌肉中的核定位。总的来说，这项研究将为EDMD的细胞病理学提供新的见解，同时揭示关于核运动的新信息，核运动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.