Analysis of nucleocytoplasmic transport in muscle disorders caused by mutations in lamins A/C

本研究方案是基于一种假设，即由于细胞因子应答转录因子的核质转运受损，导致层粘胶蛋白A/C的特异性突变导致部分脂肪营养不良综合征。由于肌生长抑制素缺乏的动物和受某些纤层蛋白突变影响的患者之间有着惊人的相似性，我们的分析将集中在肌生长抑制素/SMAD信号系统上。我们将采用已建立的分析成肌细胞和肌纤维的核细胞质运输。随后，我们将分析信号转导在健康细胞和病变细胞中的易位。除了组织细胞化学，我们还将使用共聚焦荧光显微镜，重组蛋白的显微注射，渗透细胞的体外转运试验，以及重组DNA的细胞转染。在这些实验中，我们有机会获得不朽的肌肉细胞系，正常对照个体的材料，以及具有分子特征的层压板病患者的材料。相关cdna （lamins, SMADs, STATs）可用。

This research proposal is based on the hypothesis that specific mutations in lamins A/C cause partial lipodystrophy syndromes due to the impaired nucleocytoplasmic transport of cytokine responsive transcription factors. Because of the striking similarities between myostatin deficient animals and patients affected by certain lamin mutations, our analysis will concentrate on the myostatin/SMAD signaling system. We will adopt the established analyses of nucleocytoplasmic transport for myoblasts and muscle fibers. Subsequently, we will analyze the translocation of signal transducers in healthy and diseased cells. Besides histocytochemistry, we will use confocal fluorescence microscopy, the microinjection of recombinant proteins, and in-vitro-transport assays with permeabilized cells, and the transfection of cells with recombinant DNA. For these experiments, we have access to an immortalized muscle cell line, to material from normal control individuals, and from patients with molecularly characterized laminopathies. The relevant cDNAs (lamins, SMADs, STATs) are available.