Nile red fluorescence screening facilitating neutral lipid phenotype determination in budding yeast, Saccharomyces cerevisiae, and the fission yeast Schizosaccharomyces pombe

Rostron, Kerry A; Rolph, Carole E.; Lawrence, Clare

doi:10.1007/s10482-015-0467-6

Cited by 20 publications

(11 citation statements)

References 50 publications

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“…The procedure for neutral lipid droplet quantification was adapted from [ 34 ]. Live cells growing exponentially in YES were stained with Nile red (10 μM; Sigma) for 10 min at room temperature and images were taken using the Olympus CellR system (GFP filter; excitation 475 nm, emission 530 nm).…”

Section: Methodsmentioning

confidence: 99%

Fission Yeast CSL Transcription Factors: Mapping Their Target Genes and Biological Roles

et al. 2015

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BackgroundCbf11 and Cbf12, the fission yeast CSL transcription factors, have been implicated in the regulation of cell-cycle progression, but no specific roles have been described and their target genes have been only partially mapped.Methodology/Principal FindingsUsing a combination of transcriptome profiling under various conditions and genome-wide analysis of CSL-DNA interactions, we identify genes regulated directly and indirectly by CSL proteins in fission yeast. We show that the expression of stress-response genes and genes that are expressed periodically during the cell cycle is deregulated upon genetic manipulation of cbf11 and/or cbf12. Accordingly, the coordination of mitosis and cytokinesis is perturbed in cells with genetically manipulated CSL protein levels, together with other specific defects in cell-cycle progression. Cbf11 activity is nutrient-dependent and Δcbf11-associated defects are mitigated by inactivation of the protein kinase A (Pka1) and stress-activated MAP kinase (Sty1p38) pathways. Furthermore, Cbf11 directly regulates a set of lipid metabolism genes and Δcbf11 cells feature a stark decrease in the number of storage lipid droplets.Conclusions/SignificanceOur results provide a framework for a more detailed understanding of the role of CSL proteins in the regulation of cell-cycle progression in fission yeast.

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Section: Methodsmentioning

confidence: 99%

Fission Yeast CSL Transcription Factors: Mapping Their Target Genes and Biological Roles

et al. 2015

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“… 2012 ; Meyers et al. 2017 ) and they serve as a useful proxy for monitoring lipid biosynthesis (Rostron, Rolph and Lawrence 2015 ). Storage neutral lipids can be used to produce the membrane phospholipids required during mitosis (Makarova et al.…”

Section: Resultsmentioning

confidence: 99%

Mitotic defects in fission yeast lipid metabolism ‘cut’ mutants are suppressed by ammonium chloride

Zach

Tvarůžková

Schätz

et al. 2018

FEMS Yeast Research

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Fission yeast ‘cut’ mutants show defects in temporal coordination of nuclear division with cytokinesis, resulting in aberrant mitosis and lethality. Among other causes, the ‘cut’ phenotype can be triggered by genetic or chemical perturbation of lipid metabolism, supposedly resulting in shortage of membrane phospholipids and insufficient nuclear envelope expansion during anaphase. Interestingly, penetrance of the ‘cut’ phenotype in mutants of the transcription factor cbf11 and acetyl-coenzyme A carboxylase cut6, both related to lipid metabolism, is highly dependent on growth media, although the specific nutrient(s) affecting ‘cut’ occurrence is not known. In this study, we set out to identify the growth media component(s) responsible for ‘cut’ phenotype suppression in Δcbf11 and cut6–621 cells. We show that mitotic defects occur rapidly in Δcbf11 cells upon shift from the minimal EMM medium (‘cut’ suppressing) to the complex YES medium (‘cut’ promoting). By growing cells in YES medium supplemented with individual EMM components, we identified ammonium chloride, an efficiently utilized nitrogen source, as a specific and potent suppressor of the ‘cut’ phenotype in both Δcbf11 and cut6–621. Furthermore, we found that ammonium chloride boosts lipid droplet formation in wild-type cells. Our findings suggest a possible involvement of nutrient-responsive signaling in ‘cut’ suppression.

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“…Nile red fluorescence is widely used to screen new oleaginous yeasts (Hicks et al, 2019;Sitepu et al, 2019;Zhao et al, 2019), but also to quantify neutral lipids in non-oleaginous yeasts, such as S. cerevisiae or Schizosaccharomyces pombe (Rostron et al, 2015). Other dyes are widely used to screen microalgae, such as BODIPY (Rumin et al, 2015), but still with few reports for oleaginous yeasts (for example Patel et al, 2015;Rakicka et al, 2015;Morin et al, 2020).…”

Section: Discussionmentioning

confidence: 99%

Nile Red Incubation Time Before Reading Fluorescence Greatly Influences the Yeast Neutral Lipids Quantification

et al. 2021

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High-throughput screening methodologies to estimate lipid content in oleaginous yeasts use Nile red fluorescence in a given solvent and optimized excitation/emission wavelengths. However, Nile red fluorescence stabilization has been poorly analyzed, and high variability occurs when relative fluorescence is measured immediately or a few minutes after dye addition. The aim of this work was to analyze the fluorescence of Nile red at different incubation times using a variety of solvents and oleaginous/non-oleaginous yeast strains. We showed that fluorescence stabilization occurs between 20 and 30 min, depending on the strain and solvent. Therefore, we suggest that fluorescence measurements should be followed until stabilization, where Relative Fluorescence Units should be considered after stabilization for lipid content estimation.

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Nile red fluorescence screening facilitating neutral lipid phenotype determination in budding yeast, Saccharomyces cerevisiae, and the fission yeast Schizosaccharomyces pombe

Cited by 20 publications

References 50 publications

Fission Yeast CSL Transcription Factors: Mapping Their Target Genes and Biological Roles

Fission Yeast CSL Transcription Factors: Mapping Their Target Genes and Biological Roles

Mitotic defects in fission yeast lipid metabolism ‘cut’ mutants are suppressed by ammonium chloride

Nile Red Incubation Time Before Reading Fluorescence Greatly Influences the Yeast Neutral Lipids Quantification

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