Large-scale analysis of diffusional dynamics of proteins in living yeast cells using fluorescence correlation spectroscopy
Large-scale analysis of diffusional dynamics of proteins in living yeast cells using fluorescence correlation spectroscopy
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DOI：
10.1016/j.bbrc.2019.09.066
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发表时间：
2019-12-03
影响因子：
3.1
通讯作者：
Taguchi, Hideki
中科院分区：
文献类型：
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作者：
Fukuda, Takafumi;Kawai-Noma, Shigeko;Taguchi, Hideki
在活细胞中，大多数蛋白质并不是单独发挥作用，而是与其他蛋白质或其他生物分子相互作用，维持细胞功能，构成“蛋白质群落”。先前对基于质谱的蛋白质相互作用网络（相互作用组）的研究已经提供了蛋白质群落的图景。然而，这些都是细胞被破坏后的静态信息。为了更好地了解细胞中的蛋白质群落，了解细胞内动力学和相互作用的特性非常重要。由于蛋白质的流体动力学大小和流动性与这些特性相关，因此直接测量单个活细胞中蛋白质的扩散运动将有助于揭示这些特性。在这里，我们使用荧光相关光谱 (FCS) 完成了对活酵母酿酒酵母细胞中 369 种细胞质 GFP 融合蛋白的扩散和同源寡聚特性的测量。大规模分析表明，大多数蛋白质的运动遵循二元（即慢分量和快分量）扩散模型。值得注意的是，这两种组分的扩散速度都比预期的单体状态要慢。此外，进一步的分析表明，活细胞中以同源寡聚状态存在的蛋白质比之前预期的要多。我们的研究大规模地表征了活细胞中蛋白质的动态，提供了细胞内蛋白质动态的全局视图，以了解蛋白质群落。 (C) 2019 Elsevier Inc. 保留所有权利。
In the living cells, the majority of proteins does not work alone, but interact with other proteins or other biomolecules to maintain the cellular function, constituting a "protein community". Previous efforts on mass spectroscopy-based protein interaction networks, interactomes, have provided a picture on the protein community. However, these were static information after cells were disrupted. For a better understanding of the protein community in cells, it is important to know the properties of intracellular dynamics and interactions. Since hydrodynamic size and mobility of proteins are related into such properties, direct measurement of diffusional motion of proteins in single living cells will be helpful for uncovering the properties. Here we completed measurement of the diffusion and homo-oligomeric properties of 369 cytoplasmic GFP-fusion proteins in living yeast Saccharomyces cerevisiae cells using fluorescence correlation spectroscopy (FCS). The large-scale analysis showed that the motions of majority of proteins obeyed a two-component (i.e. slow and fast components) diffusion model. Remarkably, both of the two components diffused more slowly than expected monomeric states. In addition, further analysis suggested that more proteins existed as homo-oligomeric states in living cells than previously expected. Our study, which characterizes the dynamics of proteins in living cells on a large-scale, provided a global view on intracellular protein dynamics to understand the protein community. (C) 2019 Elsevier Inc. All rights reserved.