Novel nucleolar isolation method reveals rapid response of human nucleolar proteomes to serum stimulation
| Autor: | Yun Wah Lam, Yi Min Liang, Kin Yan So, Xian Wang, Zhou Fang Li, Rajkumar Ramalingam |
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| Rok vydání: | 2012 |
| Předmět: |
Proteomics
Serum DNA Repair Proteome Nucleolus Quantitative proteomics Biophysics Nuclear Proteins Biology Cell Fractionation Biochemistry Stable isotope labeling by amino acids in cell culture Organelle Humans Proliferation Marker Ribosomes Biogenesis Cell Nucleolus Cell Proliferation DNA Damage HeLa Cells |
| Zdroj: | Journal of proteomics. 77 |
| ISSN: | 1876-7737 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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