IVEM TOMOGRAPHY OF ISOLATED EXTRACTED & RE CONSTRUCTED SPISULA CENTROSOMES: CLAM

分离提取的 IVEM 断层扫描

• 批准号: 6119667
• 负责人: ROBERT E PALAZZO
• 金额: $ 0.85万
• 依托单位: WADSWORTH CENTER
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-01-01 至 1999-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6119667
• 关键词: animal tissue; bioimaging /biomedical imaging; biomedical equipment development; biomedical resource; computers; microscopy; nucleic acids; technology /technique

(Supported by NIH GMS 43264 to R. Palazzo and GMS 40198 to C. Rieder) This is an ongoing project in which IVEM and HVEM tomography are being used to analyze the structure of centrosomes isolated from Spisula (surf clam) oocytes prior to and after various experimental manipulations. This year we characterized the high-resolution 3-D structure of control and KI extracted centrosomes, and also extracted centrosomes that had regained their function by incubation in high speed extracts from oocytes. We found that heavily KI extracted centrosomes are non-functional and consist of an insoluble 15-nm filamentous "centromatrix" material. When this centromatrix is incubated in high speed extracts of activated egg oocytes, the extracted centrosome regains its functional abilities. The loss of centrosome function is correlated, at the IVEM tomographic level, with the loss of gamma tubulin containing 20-nm diameter ring-shaped structures, while regain of function is correla ted with the reappearance of these structures. The data reveal that the centromatrix serves as a scaffold for binding those proteins involved in microtubule nucleation. Because centrosomes are extremely small (0.25 by 0.5 um) and complex, the only way their morphology and replication can be studied is with 3-D EM. Over the past 12 years we have shown that IVEM/HVEM tomography of serial thick sections greatly facilitates such studies, and have published numerous papers on this topic-many recently with the Palazzo laboratory. We are now exploring the utility of combining high resolution antibody labeling with IVEM tomography of high pressure frozen and freeze substituted samples. Schnackenberg, B.J., A. Khodjakov, C. L. Rieder and R. E. Palazzo (1998). The disassembly and reassembly of functional centrosomes in vitro. Proc. Natl. Acad. Sci. USA 95:9295-9300.

（由NIH GMS 43264支持。Palazzo和GMS 40198到C。 里德尔）这是一个正在进行的项目，其中IVEM和HVEM断层扫描 正被用来分析中心体的结构， Spisula（冲浪蛤）卵母细胞之前和之后的各种实验 操纵 今年我们将高分辨率3D 对照和KI提取的中心体的结构，还提取了 中心体通过在高浓度下孵育恢复了功能， 从卵母细胞中快速提取。 我们发现大量提取的KI 中心体是非功能性的，由不溶性的15-nm 丝状"中心基质"物质。 当这个中心矩阵 在激活的卵母细胞的高速提取物中孵育， 提取的中心体恢复其功能能力。 的损失 在IVEM断层水平，中心体功能与 含有20-nm直径的环状的γ微管蛋白的损失 结构，而功能的恢复则与 这些结构的再现。 数据显示， centromatrix作为一个支架， 在微管成核中。 因为中心体非常小 (0.25 0.5微米）和复杂，唯一的方式，他们的形态和 可以用3D EM研究复制。 在过去的12年里，我们 已经表明，IVEM/HVEM层析成像的系列厚切片大大 促进了这些研究，并发表了许多关于这方面的论文， 许多人最近在Palazzo实验室工作。 我们现在正在探索 高分辨率抗体标记与IVEM结合的效用 高压冷冻和冷冻替代样品的断层扫描。 Schnackenberg，B.J.，a.霍贾科夫角L.里德尔和R. e. Palazzo （1998年）。 功能性中心体的解体和重组 体外 Proc. Natl. Acad. sci. USA 95：9295 - 9300.