Histone Abundance Quantification via Flow Cytometry of Htb2-GFP Allows Easy Monitoring of Cell Cycle Perturbations in Living Yeast Cells, Comparable to Standard DNA Staining.

Assaying changes in the amount of DNA in single cells is a well-established method for studying the effects of various perturbations on the cell cycle. A drawback of this method is the need for a fixation procedure that does not allow for in vivo study nor simultaneous monitoring of additional parameters such as fluorescence of tagged proteins or genetically encoded indicators. In this work, we report on a method of Histone Abundance Quantification (HAQ) of live yeast harboring a GFP-tagged histone, Htb2. We show that it provides data highly congruent with DNA levels, both in Saccharomyces cerevisiae and Ogataea polymorpha yeasts. The protocol for the DNA content assay was also optimized to be suitable for both Ogataea and Saccharomyces yeasts. Using the HAQ approach, we demonstrate the expected effects on the cell cycle progression for several compounds and conditions and show usability in conjunction with additional fluorophores. Thus, our data provide a simple approach that can be utilized in a wide range of studies where the effects of various stimuli on the cell cycle need to be monitored directly in living cells.

The GEM-GECO Calcium Indicator Is Useable in Ogataea parapolymorpha Yeast, but Aggravates Effects of Increased Cytosolic Calcium Levels.

Ca2+ is a ubiquitous second messenger, which allows eukaryotic cells to respond to external stimuli. The use of genetically encoded Ca2+ indicators allows real-time monitoring of cytosolic Ca2+ levels to study such responses. Here we explored the possibility of using the ratiometric Ca2+ indicator GEM-GECO for monitoring cytosolic Ca2+ concentration ([Ca2+]cyt) in the yeast Ogataea parapolymorpha. High-level production of GEM-GECO led to a severe growth defect in cells lacking the vacuolar Ca2+ ATPase Pmc1, which is involved in [Ca2+]cyt control, and prompted a phenotype resembling that of Pmc1 deficiency, in a strain with wild-type PMC1. This was likely due to the presence of the calmodulin domain in GEM-GECO. In contrast to previous studies of genetically-encoded calcium indicators in neuronal cells, our results suggest that physiological effects of GEM-GECO expression in yeast cells are due not to Ca2+ depletion, but to excessive Ca2+ signaling. Despite these drawbacks, study of fluorescence in individual cells revealed switching of GEM-GECO from the Ca2+-free to Ca2+-bound state minutes after external addition of CaCl2. This was followed by gradual return of GEM-GECO to a Ca2+-free-state that was impaired in the pmc1-Δ mutant. These results demonstrate GEM-GECO usability for [Ca2+]cyt monitoring in budding yeast.