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

Abstract

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.

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“…This can be approached by testing new brighter fluorescent proteins, as well as those with spectra in which the background fluorescence is weaker. Another important issue is the time of incubation with the drug, because as of now, we used a six hour incubation, which can, potentially, miss transient and rapid cellular responses, such as those reported for calcium levels in yeast cells [58].…”

Fluorescent Protein-Based Sensors for Detecting Essential Metal Ions across the Tree of Life

“…106,107,133 Recently, GCaMP6s was used to image Ca 2+ fluctuations in fission yeast (Schizosaccharomyces pombe), and GEM-GECO1 was used to monitor cytosolic Ca 2+ concentrations in budding yeast (Ogataea parapolymorpha) with flow cytometry. 134,135 Although bacteria are frequently used in the screening process for the development of Ca 2+ sensors, there are few reports of their application to study Ca 2+ homeostasis in bacteria. The GCaMP6 series of sensors was sufficiently sensitive to enable recent application to single cells of Escherichia coli.…”

“…Application of fluorescent protein-based Ca 2+ sensors to eukaryotic single-cell organisms such as yeast has been significantly more limited. Both FRET and single fluorescent protein-based sensors have been applied to detect Ca 2+ bursts in Saccharomyces cerevisiae . ,, Recently, GCaMP6s was used to image Ca 2+ fluctuations in fission yeast ( Schizosaccharomyces pombe ), and GEM-GECO1 was used to monitor cytosolic Ca 2+ concentrations in budding yeast ( Ogataea parapolymorpha ) with flow cytometry. , Although bacteria are frequently used in the screening process for the development of Ca 2+ sensors, there are few reports of their application to study Ca 2+ homeostasis in bacteria. The GCaMP6 series of sensors was sufficiently sensitive to enable recent application to single cells of Escherichia coli . , Prior work relied on luminescence-based assays to measure Ca 2+ fluctuations in populations of cells.…”

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

“…To show that HAQ can be easily combined with other fluorophores, we created O. parapolymorpha strains expressing Htb2-GFP and the calcium sensor R-GECO, which exhibits red fluorescence and increased brightness at higher calcium concentrations [24]. A related green ratiometric calcium sensor, GEM-GECO, was recently tested in O. parapolymorpha by our group [25] and was shown to be functional. However, it was not suitable for our task since its fluorescence spectrum is highly similar to the GFP used to tag Htb2.…”