Mechanical slowing-down of cytoplasmic diffusion allows in vivo counting of proteins in individual cells.
Mechanical slowing-down of cytoplasmic diffusion allows in vivo counting of proteins in individual cells.
复制标题
DOI：
10.1038/ncomms11641
复制
发表时间：
2016-05-18
影响因子：
16.6
通讯作者：
Paulsson J
中科院分区：
文献类型：
--
作者：
Okumus B;Landgraf D;Lai GC;Bakshi S;Arias-Castro JC;Yildiz S;Huh D;Fernandez-Lopez R;Peterson CN;Toprak E;El Karoui M;Paulsson J
细菌中的许多关键调节蛋白的数量太少，无法用传统方法检测到，这对单细胞分析提出了特殊的挑战，因为此类蛋白对表型异质性有很大贡献。在这里，我们开发了一个基于微流体的平台，通过机械减慢低丰度蛋白质在活大肠杆菌 (E. coli) 细胞细胞质内的扩散，实现低丰度蛋白质的单分子计数。我们的技术还允许以高通量进行自动显微镜检查，对天然生理学的干扰最小，以及可行的富集/检索。我们通过分析大肠杆菌应激反应的主调节器 RpoS 通过其接头蛋白 SprE (RssB) 的控制来说明该方法。 SprE 数量的量化表明，虽然 SprE 对于 RpoS 降解是必需的，但它的表达水平低至每个细胞周期 3-4 个分子，并且 SprE 的波动在指数期近似呈泊松分布，没有爆发的迹象。 大肠杆菌 (E. coli) 蛋白质组中的几种蛋白质表达丰度过低，无法通过标准方法检测到。在这里，作者创建了一个基于微流体的平台，通过机械减慢低丰度蛋白质在活大肠杆菌中的扩散来实现单分子计数。
Many key regulatory proteins in bacteria are present in too low numbers to be detected with conventional methods, which poses a particular challenge for single-cell analyses because such proteins can contribute greatly to phenotypic heterogeneity. Here we develop a microfluidics-based platform that enables single-molecule counting of low-abundance proteins by mechanically slowing-down their diffusion within the cytoplasm of live Escherichia coli (E. coli) cells. Our technique also allows for automated microscopy at high throughput with minimal perturbation to native physiology, as well as viable enrichment/retrieval. We illustrate the method by analysing the control of the master regulator of the E. coli stress response, RpoS, by its adapter protein, SprE (RssB). Quantification of SprE numbers shows that though SprE is necessary for RpoS degradation, it is expressed at levels as low as 3–4 molecules per average cell cycle, and fluctuations in SprE are approximately Poisson distributed during exponential phase with no sign of bursting. Several proteins are expressed at too low abundance in the Escherichia coli (E. coli) proteome to be detected by standard methods. Here, the authors create a microfluidics-based platform enabling single-molecule counting of low-abundance proteins by mechanically slowing-down their diffusion in live E. coli.