Dual Color Photoactivation Localization Microscopy of Cardiomyopathy-associated Desmin Mutants
Dual Color Photoactivation Localization Microscopy of Cardiomyopathy-associated Desmin Mutants
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DOI：
10.1074/jbc.m111.313841
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发表时间：
2012-05-04
影响因子：
4.8
通讯作者：
Milting, Hendrik
中科院分区：
文献类型：
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作者：
Brodehl, Andreas;Hedde, Per Niklas;Milting, Hendrik
编码中间丝蛋白结蛋白的DES基因的突变可引起骨骼肌和心肌病，其特征通常在于结蛋白和相关蛋白在细胞水平的细胞质聚集。通过原子力显微镜，我们证明了结蛋白突变体的细丝形成缺陷，与致心律失常的右心室心肌病相关。要了解这种疾病的发病机制，它是必不可少的，以分析结蛋白丝结构的条件下，健康和突变结蛋白的表达在等摩尔水平模仿体内的情况。在这里，我们应用双色光活化定位显微镜使用光活化荧光蛋白基因融合到结蛋白和特点的杂合状态缺乏内源性结蛋白的活细胞。此外，我们应用荧光共振能量转移解开短距离的结构模式的丝结蛋白突变体。对于第一次，我们提出了一致的高分辨率数据的结构影响的五个杂合结蛋白突变在体外和活细胞中的细丝形成。我们的研究结果可能有助于从分子水平上理解心肌病中杂合DES突变的病理性肌丝形成缺陷。
Mutations in the DES gene coding for the intermediate filament protein desmin may cause skeletal and cardiac myopathies, which are frequently characterized by cytoplasmic aggregates of desmin and associated proteins at the cellular level. By atomic force microscopy, we demonstrated filament formation defects of desmin mutants, associated with arrhythmogenic right ventricular cardiomyopathy. To understand the pathogenesis of this disease, it is essential to analyze desmin filament structures under conditions in which both healthy and mutant desmin are expressed at equimolar levels mimicking an in vivo situation. Here, we applied dual color photoactivation localization microscopy using photoactivatable fluorescent proteins genetically fused to desmin and characterized the heterozygous status in living cells lacking endogenous desmin. In addition, we applied fluorescence resonance energy transfer to unravel short distance structural patterns of desmin mutants in filaments. For the first time, we present consistent high resolution data on the structural effects of five heterozygous desmin mutations on filament formation in vitro and in living cells. Our results may contribute to the molecular understanding of the pathological filament formation defects of heterozygous DES mutations in cardiomyopathies.