Inference of cell cycle-dependent proteolysis by laser scanning cytometry
Inference of cell cycle-dependent proteolysis by laser scanning cytometry
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DOI：
10.1016/j.yexcr.2009.01.011
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发表时间：
2009-06-10
影响因子：
3.7
通讯作者：
Tansey, William P.
中科院分区：
文献类型：
--
作者：
Chakraborty, Abhishek A.;Tansey, William P.
将蛋白质周转与细胞周期进程偶联的机制对于协调细胞复制和分裂事件至关重要。尽管细胞周期调节蛋白水解的重要性，然而，测量这种现象的技术是有限的，并且通常涉及监测在同步化后释放回到细胞周期的细胞。我们在这里描述了激光扫描细胞术（LSC）的使用，这是荧光显微镜和流式细胞术之间的技术合并，用于确定哺乳动物细胞未受干扰、异步培养物中蛋白质稳定性的细胞周期依赖性变化。在该方法中，LSC准确测量全细胞荧光的能力与RNA荧光原位杂交和免疫荧光一起使用，以将细胞中特定RNA和蛋白质的丰度与其在细胞周期中的点相关联。RNA和蛋白质水平的平行监测与蛋白质合成抑制剂一起使用，以揭示蛋白质周转的细胞周期特异性变化。我们证明了这种方法的可行性，通过分析两个突出的人类癌蛋白，Myc和细胞周期蛋白E的蛋白水解，并认为这种基于LSC的方法提供了几个实际的优势，传统的细胞同步化方法。(C)2009 Elsevier Inc. All rights reserved.
Mechanisms that couple protein turnover to cell cycle progression are critical for coordinating the events of cell duplication and division. Despite the importance of cell cycle-regulated proteolysis, however, technologies to measure this phenomenon are limited, and typically involve monitoring cells that are released back into the cell cycle after synchronization. We describe here the use of laser scanning cytometry (LSC), a technical merger between fluorescence microscopy and flow cytometry, to determine cell cycle-dependent changes in protein stability in unperturbed, asynchronous, cultures of mammalian cells. In this method, the ability of the LSC to accurately measure whole cell fluorescence is employed, together with RNA fluorescence in situ hybridization and immunofluorescence, to relate abundance of a particular RNA and protein in a cell to its point at the cell cycle. Parallel monitoring of RNA and protein levels is used, together with protein synthesis inhibitors, to reveal cell cycle-specific changes in protein turnover. We demonstrate the viability of this method by analyzing the proteolysis of two prominent human oncoproteins, Myc and Cyclin E, and argue that this LSC-based approach offers several practical advantages over traditional cell synchronization methods. (C) 2009 Elsevier Inc. All rights reserved.