KINETOCHORE STRUCT COMPARING CHEMICAL FIXATION W/ HIGH PRESSURE FREEZING

着丝粒结构比较化学固定与高压冷冻

• 批准号: 6119649
• 负责人: BRUCE F MCEWEN
• 金额: $ 1.99万
• 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/6119649
• 关键词: bioengineering /biomedical engineering; bioimaging /biomedical imaging; biomedical equipment development; biomedical resource; computers; microscopy; technology /technique

(Supported by the NIH/NCRR/P41 RR 01219 grant, NSF MCB9808879 to B. McEwen and NIH GMS 40198 to C. L. Rieder). Three decades of structural analyses have produced the view that the kinetochore in vertebrate cells is a disk-shaped structure composed of three distinct structural domains. The most prominent of these consists of a conspicuous electron opaque "outer plate" that is separated by a light staining electron translucent "middle" plate from an "inner" plate associated with the surface of the pericentric heterochromatin. Spindle microtubules terminate in the outer plate and, in their absence, a conspicuous "corona" of fine filaments radiates from the cytoplasmic surface of this plate. Here we report for the first time the ultrastructure of kinetochores in untreated and colcemid-treated vertebrate somatic (PtK1) cells prepared for optimal structural preservation using high-pressure freezing and freeze-substitution. In serial thin sections, and IVEM tomographic re construc tions, the kinetochore appears as a 50-75 nm thick mat of light-staining fibrous material that is directly connected with the more electron opaque surface of the centromeric heterochromatin. This mat corresponds to the outer plate in conventional preparations, and it is surrounded on its cytoplasmic surface by a conspicuous 100-150 nm wide zone that excludes all ribosomes and other cytoplasmic components. High magnification views of this zone reveal that it contains a loose network of light staining 9-nm thick fibers that are analogous to the corona fibers in conventional preparations. Unlike the chromosome arms which appear uniformly electron opaque, the chromatin in the primary constriction appears mottled. Since and electron-translucent "middle" plate is not visible in these kinetochore preparations this zone is likely an artifact produced during conventional fixation and/or dehydration procedures. McEwen, B.F., C-E.Hsieh, A. L. Mattheyses and C. L. Rieder. 1998. A new look at kinetochore structure in vertebrate somatic cells using high-pressure freezing and freeze substitution. Chromosoma (in press) McEwen, B.F., C-E. Hsieh, A. L. Mattheyses and C. L. Rieder. (1998). A new look at kinetochore structure in vertebrate somatic cells using high-pressure freezing and freeze substitution. Molecular Biology of the Cell

（由NIH/NCRR/P41 RR 01219资助，NSF MCB 9808879支持， B。McEwen和NIH GMS 40198对C. L.里德尔）。 三十年的 结构分析表明， 脊椎动物细胞是由三种不同的细胞组成的盘状结构， 结构域 其中最突出的包括一个 一个明显的电子不透明的“外板”，由一盏灯隔开， 从"内"板染色电子半透明的"中"板 与臂间异染色质表面相关。 纺锤体微管终止于外板， 没有，一个明显的"冠"的细丝辐射从 细胞质表面。 在这里我们首次报道 未处理和秋水仙胺处理小鼠动粒超微结构 制备脊椎动物体细胞（PtK1），以获得最佳结构 使用高压冷冻和冷冻置换进行保存。 在 系列薄切片和IVEM层析成像， 动粒呈现为50 - 75 nm厚的浅色纤维垫 直接与电子不透明的材料连接的材料 着丝粒异染色质的表面。 这个垫子对应于 在传统的制备中，外板被包围在 其细胞质表面具有明显的100 - 150 nm宽的区域， 排除所有核糖体和其他细胞质成分。 高 这一区域的放大视图显示，它包含一个松散的 光染色9 nm厚的纤维网络，类似于 冠状纤维在常规制剂中。 不像染色体 臂出现均匀的电子不透明，染色质在 主缢痕有斑点。 由于和电子半透明 "中间"板在这些动粒制剂中不可见， 区域可能是传统固定过程中产生的伪影 和/或脱水过程。 McEwen，B.F.，谢世荣，A. L. Mattheyses和C. L.里德尔。 1998. 动粒的新认识 利用高压冷冻技术研究脊椎动物体细胞的结构， 冻结替代。 Chromosoma（in press）McEwen，B.F.，C-E 谢家华， a. L. Mattheyses和C. L.里德尔。 （1998年）。 动粒的新认识 利用高压冷冻技术研究脊椎动物体细胞的结构， 冻结替代。 Molecular Biology of the Cell