3-DIMENSIONAL STRUCTURAL CHARACTERIZATION OF CENTROSOMES FROM EARLY DROSOPHILA EMBRYOS
3-DIMENSIONAL STRUCTURAL CHARACTERIZATION OF CENTROSOMES FROM EARLY DROSOPHILA EMBRYOS
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DOI：
10.1083/jcb.130.5.1149
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发表时间：
1995-09-01
影响因子：
7.8
通讯作者：
AGARD, DA
中科院分区：
文献类型：
--
作者：
MORITZ, M;BRAUNFELD, MB;AGARD, DA
对中心体中微管成核位点的机制和结构的理解一直是难以捉摸的。这部分是由于难以获得大量的中心体进行分析，以及使用传统的EM技术获得可解释的结构数据的问题。我们描述了一种从早期果蝇胚胎中分离大量功能中心体的方案。利用自动电子断层扫描，我们已经开始对这些完整的中心体进行三维结构表征，这些中心体有或没有再生的mt。中心体的重建具有类似6-8 nm的分辨率，显示在mt的负端没有大的结构，这表明如果mt成核材料物理接触mt，它必须与负端形状密切一致。虽然许多MT起源于中心粒附近，但在整个PCM中发现了MT负端，甚至靠近其外边界。MTs在PCM上纵横交错，表明成核位点在许多不同的方向上定向。没有MT的中心体的重建表明，在MT再生时，PCM发生了重组；此外，在没有mt的中心体的PCM中，可以看到与mt直径相似的环状结构，可能是mt成核位点。
An understanding of the mechanism and structure of microtubule (MT)-nucleating sites within the pericentriolar material (PCM) of the centrosome has been elusive. This is partly due to the difficulty in obtaining large quantities of centrosomes for analysis, as well as to the problem of attaining interpretable structural data with conventional EM techniques, We describe a protocol for isolating a large quantity of functional centrosomes from early Drosophila embryos. Using automated electron tomography, we have begun a three-dimensional structural characterization of these intact centrosomes with and without regrown MTs. Reconstructions of the centrosomes to similar to 6-8 nm resolution revealed no large structures at the minus ends of MTs, suggesting that if MT-nucleating material physically contacts the MTs, it must conform closely to the shape of the minus end. While many MTs originate near the centrioles, MT minus ends were found throughout the PCM, and even close to its outer boundary. The MTs criss-crossed the PCM, suggesting that nucleating sites are oriented in many different directions. Reconstructions of centrosomes without MTs suggest that there is a reorganization of the PCM upon MT regrowth; moreover, ring-like structures that have a similar diameter as MTs are apparent in the PCM of centrosomes without MTs, and may be MT-nucleating sites.