PDF(Pubmed)
Abstract：
The measurement of dynamic changes in protein level and localization throughout the cell cycle is of major relevance to studies of cellular processes tightly coordinated with the cycle, such as replication, transcription, DNA repair, and checkpoint control. Currently available methods include biochemical assays of cells in bulk following synchronization, which determine protein levels with poor temporal and no spatial resolution. Taking advantage of genetic engineering and live-cell microscopy, we performed time-lapse imaging of cells expressing fluorescently tagged proteins under the control of their endogenous regulatory elements in order to follow their levels throughout the cell cycle. We effectively discern between cell cycle phases and S subphases based on fluorescence intensity and distribution of co-expressed proliferating cell nuclear antigen (PCNA)-mCherry. This allowed us to precisely determine and compare the levels and distribution of multiple replication-associated factors, including Rap1-interacting factor 1 (RIF1), minichromosome maintenance complex component 6 (MCM6), origin recognition complex subunit 1 (ORC1, and Claspin, with high spatiotemporal resolution in HeLa Kyoto cells. Combining these data with available mass spectrometry-based measurements of protein concentrations reveals the changes in the concentration of these proteins throughout the cell cycle. Our approach provides a practical basis for a detailed interrogation of protein dynamics in the context of the cell cycle.
摘要：
测量蛋白质水平的动态变化和整个细胞周期的定位与研究与周期紧密协调的细胞过程具有重要意义。比如复制，转录,DNA修复,和检查站控制。目前可用的方法包括同步后大量细胞的生化测定，确定蛋白质水平的时间差，没有空间分辨率。利用基因工程和活细胞显微镜，我们在内源性调节元件的控制下对表达荧光标记蛋白的细胞进行延时成像,以跟踪它们在整个细胞周期中的水平.我们基于共表达的增殖细胞核抗原（PCNA）-mCherry的荧光强度和分布，有效地辨别细胞周期阶段和S亚期。这使我们能够精确地确定和比较多个复制相关因子的水平和分布，包括Rap1相互作用因子1(RIF1)，微型染色体维持复合物组件6（MCM6），起源识别复合物亚基1(ORC1和Claspin，在HeLa京都细胞中具有较高的时空分辨率。将这些数据与可用的基于质谱的蛋白质浓度测量相结合，揭示了这些蛋白质在整个细胞周期中的浓度变化。我们的方法为在细胞周期背景下详细询问蛋白质动力学提供了实践基础。
