Analysis of cryo-electron microscopy images does not support the existence of 30-nm chromatin fibers in mitotic chromosomes in situ
Analysis of cryo-electron microscopy images does not support the existence of 30-nm chromatin fibers in mitotic chromosomes in situ
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DOI：
10.1073/pnas.0810057105
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发表时间：
2008-12-16
影响因子：
11.1
通讯作者：
Dubochet, Jacques
中科院分区：
文献类型：
--
作者：
Eltsov, Mikhail;MacLellan, Kirsty M.;Dubochet, Jacques
尽管30 nm染色质纤维的形成被认为是染色质紧凑的最基本事件，但它仍然存在争议，因为直到现在才有可能在活的真核细胞中对染色质进行高分辨率成像。玻璃体切片的冷冻电子显微镜是一项相对较新的技术，它能够直接高分辨率地观察接近自然状态的细胞结构。我们使用冷冻电子显微镜和图像处理技术进一步研究了人类有丝分裂染色体中30 nm染色质纤维的存在。用高压冷冻法玻璃化冷冻HeLa S3细胞，薄片，不经进一步化学处理或染色，在冷冻电子显微镜下成像。为了对图像进行明确的解释，对比度传递函数的影响进行了计算校正。HeLa S3细胞的有丝分裂染色体结构致密，纹理均匀，无明显的30 nm纤维。染色体图像的功率谱也没有显示30 nm染色质折叠的迹象。这些结果，加上我们对染色体膨胀效应的观察，强烈表明，在紧凑的中期染色体的大部分中，核小体纤维不经历30 nm的折叠，而是以高度无序和交错的状态存在，在局部尺度上，这与聚合物熔体相当。
Although the formation of 30-nm chromatin fibers is thought to be the most basic event of chromatin compaction, it remains controversial because high-resolution imaging of chromatin in living eukaryotic cells had not been possible until now. Cryo-electron microscopy of vitreous sections is a relatively new technique, which enables direct high-resolution observation of the cell structures in a close-to-native state. We used cryo-electron microscopy and image processing to further investigate the presence of 30-nm chromatin fibers in human mitotic chromosomes. HeLa S3 cells were vitrified by high-pressure freezing, thin-sectioned, and then imaged under the cryo-electron microscope without any further chemical treatment or staining. For an unambiguous interpretation of the images, the effects of the contrast transfer function were computationally corrected. The mitotic chromosomes of the HeLa S3 cells appeared as compact structures with a homogeneous grainy texture, in which there were no visible 30-nm fibers. Power spectra of the chromosome images also gave no indication of 30-nm chromatin folding. These results, together with our observations of the effects of chromosome swelling, strongly suggest that, within the bulk of compact metaphase chromosomes, the nucleosomal fiber does not undergo 30-nm folding, but exists in a highly disordered and interdigitated state, which is, on the local scale, comparable with a polymer melt.