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: 6220675
• 负责人: YASOHIRO IKEDA
• 金额: $ 3.85万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-05-15 至 2000-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6220675
• 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.

核周间隔(PNC)是一种独特的核结构 优先定位在核仁的外围，并且是 与转化的细胞有关，包括几个人类癌细胞 台词。由RNA聚合酶III转录的几个小RNA和 HnRNP蛋白，即多嘧啶结合蛋白(PTB)，具有 已在PNC中本地化。使用GFP-PTB融合蛋白进行后续研究 在活细胞中，我们发现PNC是一个动态的 结构帽在和的外围作小的移动 随着时间的推移偶尔会进入核仁。在超微结构 水平，使用抗PTB抗体和曙红荧光 光氧化时，我们观察到PNC与 核仁的表面，有时延伸到核仁的 核仁本身。结构高度不规则，不清楚 在PNC和其余部分之间观察到了构造边界 核质。从1.5米厚的切片上查看的立体图像 IV-EM显示PNC内有链状结构。高分辨率 使用GFP-PTB对未染色的PNC进行成像 荧光法识别PNC在光线下的位置 微观层面。对转基因细胞进行拍照，最理想的是 用于电子显微镜检查的固定和加工。核电 与PNC相对应的区域似乎是电子致密的 由约80-180纳米英寸的粗、短链组成的结构 戴米特。每条链都被电子密度较低的区域所包围。 其中一些链直接连接到核仁，并可能 在单独的部分相当长，可达15米长。我们试图 确定这些粗股是否相互连接，以及它们是否形成 高阶结构。要解决PNC的3D组织问题，请访问 最佳固定HeLa连续薄切片中的高分辨率PNC 用电子显微镜观察细胞核。立体式 重建显示，PNC是一个高度相互联系和 与核仁表面的一部分相关的网状网状物。 然而，在形状、大小和形状上有相当大的差异 PNC与核仁的关系。在一些单元格中，PNC 主要位于核仁的表面，而在 在其他情况下，PNC像塞子一样伸入核仁。 有时，核仁的一部分延伸到PNC内。这 可变性可以代表动态亚核的冻结帧 结构。这些3D重建显示，PNC是 在结构上与核仁不同，形成网状网状结构 与核仁表面相关。总而言之，这些发现 提示PNC可能是一个与RNA有关的功能区块 转化细胞在细胞核内的代谢。这项工作是 发表在《细胞生物学杂志》(Huang et al.，J.Cell. 生物，137：965-974,1997)，第二个手稿正在准备中。