Department of Biology and Chemistry, City University of Hong Kong, Hong Kong, China.
J Proteomics. 2012 Dec 21;77:521-30. doi: 10.1016/j.jprot.2012.09.031. Epub 2012 Oct 31.
The nucleolus is the location of ribosomal biogenesis, and plays crucial regulatory roles in nuclear responses to stress. Here, we report a new and improved nucleolar isolation method, which is simpler and more efficient than the traditional method. The purity of nucleoli obtained by using the new protocol is comparable to that by using the classical method, as judged by electron microscopy, Western blotting and SILAC-based quantitative proteomics. Moreover, the improved efficiency of cell harvesting in the new method, biochemical events in the nucleolus could be "frozen" and captured at precisely controlled time points. Time-lapse nucleolar proteomics after serum stimulation in HeLa cell revealed for the first time that some nucleolar proteins respond to serum stimulation within a time period as short as the first 5 min of serum re-stimulation. Proteins involved in ribosomal biogenesis and in DNA damage repair are among the most dynamic proteins during the first 10 min after serum replenishment. Notably, the proliferation marker Ki-67 is also found to enter the nucleolus after serum replenishment. To our knowledge, this is the first study that demonstrates such fast responses in the nucleolus, further confirming the rapid plasticity of this organelle.
核仁是核糖体生物发生的位置，在核对应激的反应中起着至关重要的调节作用。在这里，我们报告了一种新的、改进的核仁分离方法，该方法比传统方法更简单、更高效。使用新方案获得的核仁的纯度与使用经典方法获得的核仁的纯度相当，这可以通过电子显微镜、Western blot 和 SILAC 定量蛋白质组学来判断。此外，新方法中细胞收获效率的提高，可以在精确控制的时间点“冻结”和捕获核仁中的生化事件。在 HeLa 细胞中用血清刺激进行的核仁蛋白质组学时间推移研究首次表明，一些核仁蛋白在血清再刺激后的第一个 5 分钟内就对血清刺激有反应。在血清补充后的前 10 分钟内，核糖体生物发生和 DNA 损伤修复相关的蛋白是最具动态性的蛋白之一。值得注意的是，增殖标志物 Ki-67 在血清补充后也被发现进入核仁。据我们所知，这是首次在核仁中观察到如此快速的反应，进一步证实了该细胞器的快速可塑性。
