Valli Minoska, Sauer Michael, Branduardi Paola, Borth Nicole, Porro Danilo, Mattanovich Diethard
Institute of Applied Microbiology, University of Natural Resources and Applied Life Sciences, Muthgasse 18, A-1190 Vienna, Austria.
Appl Environ Microbiol. 2005 Mar;71(3):1515-21. doi: 10.1128/AEM.71.3.1515-1521.2005.
Intracellular pH has an important role in the maintenance of the normal functions of yeast cells. The ability of the cell to maintain this pH homeostasis also in response to environmental changes has gained more and more interest in both basic and applied research. In this study we describe a protocol which allows the rapid determination of the intracellular pH of Saccharomyces cerevisiae cells. The method is based on flow cytometry and employs the pH-dependent fluorescent probe carboxy SNARF-4F. The protocol attempts to minimize the perturbation of the system under study, thus leading to accurate information about the physiological state of the single cell. Moreover, statistical analysis performed on major factors that may influence the final determination supported the validity of the optimized protocol. The protocol was used to investigate the effect of external pH on S. cerevisiae cells incubated in buffer. The results obtained showed that stationary cells are better able than exponentially grown cells to maintain their intracellular pH homeostasis independently of external pH changes. Furthermore, analysis of the intracellular pH distribution within the cell populations highlighted the presence of subpopulations characterized by different intracellular pH values. Notably, a different behavior was observed for exponentially grown and stationary cells in terms of the appearance and development of these subpopulations as a response to a changing external pH.
细胞内pH值在维持酵母细胞正常功能方面发挥着重要作用。细胞在响应环境变化时维持这种pH稳态的能力在基础研究和应用研究中都越来越受到关注。在本研究中，我们描述了一种能够快速测定酿酒酵母细胞内pH值的方法。该方法基于流式细胞术，并使用了pH依赖性荧光探针羧基SNARF-4F。该方法试图将所研究系统的扰动降至最低，从而获得有关单细胞生理状态的准确信息。此外，对可能影响最终测定的主要因素进行的统计分析支持了优化方法的有效性。该方法用于研究外部pH值对缓冲液中培养的酿酒酵母细胞的影响。所得结果表明，与指数生长期的细胞相比，静止期细胞在外部pH值变化时更能维持其细胞内pH稳态。此外，对细胞群体内细胞内pH分布的分析突出了存在以不同细胞内pH值为特征的亚群。值得注意的是，就这些亚群作为对外部pH变化的响应的出现和发展而言，指数生长期细胞和静止期细胞表现出不同的行为。
