Noninvasive High-Throughput Single-Cell Analysis of the Intracellular pH of Saccharomyces cerevisiae by Ratiometric Flow Cytometry

Abstract

The ability of cells to maintain pH homeostasis in response to environmental changes has elicited interest in basic and applied research and has prompted the development of methods for intracellular pH measurements. Many traditional methods provide information at population level and thus the average values of the studied cell physiological phenomena, excluding the fact that cell cultures are very heterogeneous. Single-cell analysis, on the other hand, offers more detailed insight into population variability, thereby facilitating a considerably deeper understanding of cell physiology. Although microscopy methods can address this issue, they suffer from limitations in terms of the small number of individual cells that can be studied and complicated image processing. We developed a noninvasive high-throughput method that employs flow cytometry to analyze large populations of cells that express pHluorin, a genetically encoded ratiometric fluorescent probe that is sensitive to pH. The method described here enables measurement of the intracellular pH of single cells with high sensitivity and speed, which is a clear improvement compared to previously published methods that either require pretreatment of the cells, measure cell populations, or require complex data analysis. The ratios of fluorescence intensities, which correlate to the intracellular pH, are independent of the expression levels of the pH probe, making the use of transiently or extrachromosomally expressed probes possible. We conducted an experiment on the kinetics of the pH homeostasis of Saccharomyces cerevisiae cultures grown to a stationary phase after ethanol or glucose addition and after exposure to weak acid stress and glucose pulse. Minor populations with pH homeostasis behaving differently upon treatments were identified.

pH determines the energetic efficiency of the cyanobacterial CO 2 concentrating mechanism

“…All cells regulate their cytosolic pH due to the inherent pH dependence of biochemical reactions (27). However, cytosolic pH varies substantially between organisms and growth conditions (28,29). Here, we use a mathematical model to examine how the cyanobacterial CCM functions over a range of cytosolic pH values.…”

Illumination of the Spatial Order of Intracellular pH by Genetically Encoded pH-Sensitive Sensors

“…For the dual excitation of all pHluorin-based ratiometric pH probes, a flow cytometer equipped with split optics for 405- and 488-nm light paths is necessary [31,55] (Figure 4). After excitation with 488-nm and 405-nm laser light, green fluorescence is detected in separate channels, and the ratios of two fluorescence parameters ( i.e.…”

Phylogeny Poorly Predicts the Utility of a Challenging Horizontally Transferred Gene in Methylobacterium Strains

“…We therefore sought to measure how the transconjugant strains responded to the dehalogenation of DCM. We used a fluorescent biosensor to measure internal pH on a rapid, approximately 5-s time scale (44). When this biosensor was coexpressed with DcmA, we could measure the change in internal pH upon addition of DCM ( Fig.…”