Formation of Hirano bodies after inducible expression of a modified form of an actin-cross-linking protein.
Formation of Hirano bodies after inducible expression of a modified form of an actin-cross-linking protein.
复制标题
肌动蛋白交联蛋白修饰形式的诱导表达后平野体的形成。
DOI：
10.1128/ec.00379-08
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发表时间：
2009
期刊：
影响因子：
--
通讯作者：
Maselli,Andrew
中科院分区：
文献类型：
--
作者：
Reyes,JuanF;Stone,Karen;Ramos,Jeanie;Maselli,Andrew
平野小体是主要由肌动蛋白和肌动蛋白相关蛋白组成的细胞质内含物。据报道，在各种神经退行性疾病期间，包括阿尔茨海默病和肌萎缩侧索硬化症，平野小体的形成。尽管导致大脑中形成这些包涵体的潜在分子机制尚不清楚，但盘基网柄菌内源性 34-kDa 肌动蛋白结合蛋白的 C 末端片段 (CT)（氨基酸 124 至 295）的表达导致体内肌动蛋白包涵体的形成。在当前的研究中，我们报告了诱导表达系统的开发，以使用诱导启动子系统（rnrB）研究平野体形成的早期阶段。通过将 CT 与绿色荧光蛋白 (CT-GFP) 融合，我们通过荧光显微镜、流式细胞术和蛋白质印迹分析监测蛋白质表达和定位。我们观察到 CT-GFP 载体系统诱导后形成的内含物数量和大小有所增加。延时显微镜研究表明，CT-GFP 焦点与细胞皮层相关并融合形成单个大聚集体。透射电子显微镜进一步表明，这些内含物具有高度有序的超微结构，这是在各种神经退行性疾病患者死后脑样本中观察到的平野小体的病理特征。总的来说，该系统提供了一种可视化和表征真核模型中早期肌动蛋白包涵体形成事件的方法。
Hirano bodies are cytoplasmic inclusions composed mainly of actin and actin-associated proteins. The formation of Hirano bodies during various neurodegenerative disorders, including Alzheimer's disease and amyotrophic lateral sclerosis, has been reported. Although the underlying molecular mechanisms that lead to the formation of these inclusions in the brain are not known, expression of the C-terminal fragment (CT) (amino acids 124 to 295) from the endogenous 34-kDa actin-binding protein ofDictyostelium discoideumleads to the formation of actin inclusions in vivo. In the current study, we report the development of an inducible expression system to study the early phases of Hirano body formation using an inducible promoter system (rnrB). By fusing the CT to a green fluorescent protein (CT-GFP), we monitored protein expression and localization by fluorescence microscopy, flow cytometry, and Western blot analysis. We observed an increase in the number and size of inclusions formed following induction of the CT-GFP vector system. Time-lapse microscopy studies revealed that the CT-GFP foci associated with the cell cortex and fused to form a single large aggregate. Transmission electron microscopy further demonstrates that these inclusions have a highly ordered ultrastructure, a pathological hallmark of Hirano bodies observed in postmortem brain samples from patients with various neurodegenerative disorders. Collectively, this system provides a method to visualize and characterize the events that surround early actin inclusion formation in a eukaryotic model.