Three-dimensional organization of pKi-67: a comparative fluorescence and electron tomography study using fluoronanogold
Three-dimensional organization of pKi-67: a comparative fluorescence and electron tomography study using fluoronanogold
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DOI：
10.1177/002215540305101102
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发表时间：
2003-11-01
影响因子：
3.2
通讯作者：
Ploton, D
中科院分区：
文献类型：
--
作者：
Cheutin, T;O'Donohue, MF;Ploton, D
单克隆抗体 (MAb) Ki-67 通常用于临床研究来估计肿瘤的生长分数。然而，pKi-67（Ki-67 MAb 检测到的蛋白质）的作用仍然难以捉摸，尽管一些生化数据强烈表明它可能组织染色质。为了更好地了解 pKi-67 的功能组织，我们通过共聚焦显微镜和电子断层扫描研究了其在间期细胞中的三维分布。 FluoroNanogold 是一种结合致密标记物和荧光染料的单一探针，用于在光学和超微结构水平上研究 pKi-67 组织。共焦显微镜观察和 3D 重建表明 pKi-67 在核仁周围形成壳。双标记实验表明 pKi-67 与核周异染色质共定位。电子显微镜研究证实了这种密切关联，并证明 pKi-67 既不位于核仁的纤维状成分中，也不位于核仁的颗粒状成分中。最后，电子断层扫描空间分析表明，pKi-67 形成直径为 250-300 nm 的绳索，其本身由 30-50 nm 厚的纤维组成。这些详细的原位比较分析强烈表明 pKi-67 参与核仁周围染色质的高级组织。
The monoclonal antibody (MAb) Ki-67 is routinely used in clinical studies to estimate the growth fraction of tumors. However, the role of pKi-67, the protein detected by the Ki-67 MAb, remains elusive, although some biochemical data strongly suggest that it might organize chromatin. To better understand the functional organization of pKi-67, we studied its three-dimensional distribution in interphase cells by confocal microscopy and electron tomography. FluoroNanogold, a single probe combining a dense marker with a fluorescent dye, was used to investigate pKi-67 organization at the optical and ultrastructural levels. Observation by confocal microscopy followed by 3D reconstruction showed that pKi-67 forms a shell around the nucleoli. Double labeling experiments revealed that pKi-67 co-localizes with perinucleolar heterochromatin. Electron microscopy studies confirmed this close association and demonstrated that pKi-67 is located neither in the fibrillar nor in the granular components of the nucleolus. Finally, spatial analyses by electron tomography showed that pKi-67 forms cords 250-300 nm in diameter, which are themselves composed of 30-50-nm-thick fibers. These detailed comparative in situ analyses strongly suggest the involvement of pKi-67 in the higher-order organization of perinucleolar chromatin.