Jackson D A, Cook P R
Sir William Dunn School of Pathology, Oxford UK.
EMBO J. 1988 Dec 1;7(12):3667-77. doi: 10.1002/j.1460-2075.1988.tb03248.x.
Whether nucleoskeletons seen after extracting cells are preparative artefacts is controversial. Using an extraction method that preserves vital nuclear functions, we have visualized part of a nucleoskeleton by electron microscopy of thick resinless sections. Cells encapsulated in agarose microbeads are lysed using Triton in a physiological buffer; the agarose coat prevents aggregation and protects fragile cell contents. These extracted cells are accessible to small molecules and transcribe and replicate at rates close to those in vivo. After electroeluting most chromatin after treatment with HaeIII, a skeleton is uncovered which ramifies throughout the nucleus. Individual filaments are approximately 10 nm wide with an axial repeat of 23 nm, characteristic of intermediate filaments.
提取细胞后所观察到的核骨架是否为制备过程中的假象存在争议。通过使用一种能保留重要核功能的提取方法,我们利用无树脂厚切片的电子显微镜观察到了部分核骨架。包裹在琼脂糖微珠中的细胞在生理缓冲液中用Triton裂解;琼脂糖外壳可防止聚集并保护脆弱的细胞内容物。这些提取后的细胞对小分子具有通透性,并且转录和复制的速率与体内相近。在用HaeIII处理后电洗脱大部分染色质后,会暴露出一个贯穿整个细胞核分支的骨架。单个细丝的宽度约为10纳米,轴向重复距离为23纳米,这是中间丝的特征。