ABNORMAL DYSTROPHIN DISTRIBUTION IN CARDIOMYOPATHIC HAMSTER

心肌病仓鼠中肌营养不良蛋白分布异常

• 批准号: 6469036
• 负责人: YASOHIRO IKEDA
• 金额: $ 10.66万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-05-01 至 2002-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6469036
• 关键词: animal tissue; biomedical resource; biotechnology; growth /development; human tissue; microscopy; technology /technique

The perinucleolar compartment (PNC) is a unique nuclear structure preferentially localized at the periphery of the nucleolus and is associated with transformed cells, including several human cancer cell lines. Several small RNAs transcribed by RNA polymerase III and the hnRNP protein, the polypyrimidine tract binding (PTB) protein, have been localized in the PNC. Using a GFP-PTB fusion protein to follow the PNC in living cells, we showed that the PNC was a dynamic structure hat makes small movements at the periphery of and occasionally into the nucleolus over time. At the ultrastructural level, using anti-PTB antibodies and eosin fluorescence photooxidation, we observed that the PNCs are in direct contact with the surface of the nucleolus and occasionally extend into the nucleolus itself. The structure is highly irregular and no clear structural boundary was observed between the PNC and the remainder of the nucleoplasm. Stereo images from 1 5m thick sections viewed with IV EM revealed strand-like structures across the PNC. High resolution imaging of unstained PNCs was accomplished by using the GFP-PTB fluorescence to identify the locations of PNCs at the light microscopic level. Transfected cells were photographed, optimally fixed and processed for electron microscopic examination. The nuclear region corresponding to the PNCs appeared to be an electron-dense structure composed of thick, short strands measuring ~80-180 nm in daimeter. Each strand was surrounded by less electron-dense areas. Some of the strands were directly linked to the nucleolus and could be quite long in individual sections, up to 1 5m in length. We sought to determine if these thick strands are interconnected and if they form a higher order structure. To resolve the 3D organization of the PNC at high resolution, PNCs in serial thin sections of optimally fixed HeLa cell nuclei were examined by electron microscopy. Three-dimensional reconstructions revealed that the PNC is a highly interconnected and reticulated mesh associated with a portion of the nucleolar surface. However, there was considerable variability in the shape, size and the relationship of the PNC with the nucleolus. In some cells, the PNC was mostly positioned on the surface of the nucleolus, while in others, the PNC extended into the nucleolus like a plug. Occasionally, a portion of the nucleolus extended into the PNC. This variability may represent freeze frames of a dynamic subnuclear structure. These 3D reconstructions have shown that the PNC is structurally distinct from the nucleolus and forms a reticulated mesh associated with the nucleolar surface. Together, these findings suggest that the PNC may be a functional compartment involved in RNA metabolism in the nucleus of transformed cells. This work was published in the Journal of Cell Biology (Huang et al., J. Cell. Biol., 137: 965-974, 1997) and a second manuscript is in preparation.

核仁周室是一种独特的核结构 优先定位在核仁的周边， 与转化细胞相关，包括几种人类癌细胞 线 RNA聚合酶III转录的几种小RNA， hnRNP蛋白，即多聚嘧啶束结合（PTB）蛋白，具有 在PNC中被定位。 使用GFP-PTB融合蛋白来跟踪 活细胞中的PNC，我们表明PNC是一个动态的 结构帽使小的运动在外围和 偶尔会进入细胞核 在超微结构 水平，使用抗PTB抗体和曙红荧光 光氧化，我们观察到PNC与 核仁的表面，偶尔延伸到 核仁本身。 结构高度不规则， 在PNC和其余部分之间观察到结构边界， 核质 从15 m厚的切片观察的立体图像， IV EM显示跨PNC的股样结构。 高分辨率 使用GFP-PTB对未染色的PNC进行成像 荧光来识别PNC在光下的位置 微观水平。 对转染的细胞进行拍照，最佳条件是 固定并处理用于电子显微镜检查。 核 对应于PNC的区域似乎是电子致密的 由厚的短链组成的结构，尺寸约为80-180 nm， 直径计 每一条链都被电子密度较低的区域所包围。 一些链直接连接到核仁， 在个别部分相当长，长达15米。 我们试图 确定这些粗股是否相互连接， 高阶结构。 要解决PNC的3D组织，请执行以下操作： 最佳固定HeLa连续薄切片中的高分辨率PNC 用电子显微镜检查细胞核。 三维 重建显示，PNC是一个高度相互关联的， 与核仁表面的一部分相连的网状网。 然而，在形状、大小和形状上存在相当大的变化。 PNC与核仁的关系。 在一些细胞中，PNC 主要位于核仁的表面，而在 有的PNC呈塞子状伸入核仁。 偶尔有一部分核仁伸入PNC。 这 可变性可以表示动态亚核的冻结帧， 结构 这些3D重建表明，PNC是 在结构上不同于核仁并形成网状网 与核仁表面相连。 总之，这些发现 表明PNC可能是参与RNA的功能区室， 在转化细胞的细胞核中的代谢。 这项工作是 发表在《细胞生物学杂志》上（Huang等人，J.细胞。 生物学：137：965-974，1997），并且第二手稿正在准备中。