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Recapitulation of the nuclear auxin response pathway in Saccharomyces cerevisiae (yeast) provides a means to functionally assay the contribution of individual signaling components to response dynamics. Here, we describe a time course assay for characterizing auxin response circuits using flow cytometry. This method allows for quantitative measurements of the dynamic response of up to 12 circuits (strains) at once. We also describe a steady-state assay and how to utilize an R package we developed to facilitate data analysis.
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Quantifying turgor pressure in budding and fission yeasts based upon osmotic properties
Walled cells, such as plants, fungi, and bacteria cells, possess a high internal hydrostatic pressure, termed turgor pressure, that drives volume growth and contributes to cell shape determination. Rigorous measurement of turgor pressure, however, remains challenging, and reliable quantitative measurements, even in budding yeast are still lacking. Here, we present a simple and robust experimental approach to access turgor pressure in yeasts based upon the determination of isotonic concentration using protoplasts as osmometers. We propose three methods to identify the isotonic condition – three-dimensional cell volume, cytoplasmic fluorophore intensity, and mobility of a cytGEMs nano-rheology probe – that all yield consistent values. Our results provide turgor pressure estimates of 1.0 ± 0.1 MPa for Schizosaccharomyces pombe, 0.49 ± 0.01 MPa for Schizosaccharomyces japonicus, 0.5 ± 0.1 MPa for Saccharomyces cerevisiae W303a and 0.31 ± 0.03 MPa for Saccharomyces cerevisiae BY4741. Large differences in turgor pressure and nano-rheology measurements between the Saccharomyces cerevisiae strains demonstrate how fundamental biophysical parameters can vary even among wild-type strains of the same species. These side-by-side measurements of turgor pressure in multiple yeast species provide critical values for quantitative studies on cellular mechanics and comparative evolution.
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- Award ID(s):
- 2213582
- PAR ID:
- 10539405
- Editor(s):
- Martin, Sophie
- Publisher / Repository:
- Molecular Biology of the Cell
- Date Published:
- Journal Name:
- Molecular Biology of the Cell
- Volume:
- 34
- Issue:
- 13
- ISSN:
- 1059-1524
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1091/mbc.E23-06-0215
