Antonie van Leeuwenhoek

, Volume 108, Issue 1, pp 97–106 | Cite as

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

  • Kerry A. Rostron
  • Carole E. Rolph
  • Clare L. Lawrence
Original Paper

Abstract

Investigation of yeast neutral lipid accumulation is important for biotechnology and also for modelling aberrant lipid metabolism in human disease. The Nile red (NR) method has been extensively utilised to determine lipid phenotypes of yeast cells via microscopic means. NR assays have been used to differentiate lipid accumulation and relative amounts of lipid in oleaginous species but have not been thoroughly validated for phenotype determination arising from genetic modification. A modified NR assay, first described by Sitepu et al. (J Microbiol Methods 91:321–328, 2012), was able to detect neutral lipid changes in Saccharomyces cerevisiae deletion mutants with sensitivity similar to more advanced methodology. We have also be able to, for the first time, successfully apply the NR assay to the well characterised fission yeast Schizosaccharomyces pombe, an increasingly important organism in biotechnology. The described NR fluorescence assay is suitable for increased throughput and rapid screening of genetically modified strains in both the biotechnology industry and for modelling ectopic lipid production for a variety of human diseases. This ultimately negates the need for labour intensive and time consuming lipid analyses of samples that may not yield a desirable lipid phenotype, whilst genetic modifications impacting significantly on the cellular lipid phenotype can be further promoted for more in depth analyses.

Keywords

Nile red Schizosaccharomyces pombe Saccharomyces cerevisiae Neutral lipids Phenotype 

Notes

Acknowledgments

This work is supported by the University of Central Lancashire and Brain Tumour North West. We acknowledge Bioneer Corporation and NBRP of the MEXT, Japan for providing deletion strains used in this study. We thank Dr. Vicky Jones and Dr. Gail Welsby for support with microscopy, Mr. Tony Dickson for technical assistance, Dr. Christopher Smith for support with the statistical analysis and Dr. Stephen Lawrence for critically reading the manuscript.

Conflict of interest

None.

References

  1. Adeyo O, Horn PJ, Lee S, Binns DD, Chandrahas A, Chapman KD, Goodman JM (2011) The yeast lipin orthologue Pah1p is important for biogenesis of lipid droplets. J Cell Biol 192:1043–1055PubMedCentralPubMedCrossRefGoogle Scholar
  2. Beller M, Thiel K, Thul PJ, Jäckle H (2010) Lipid droplets: a dynamic organelle moves into focus. FEBS Lett 584:2176–2182PubMedCrossRefGoogle Scholar
  3. Beopoulos A, Mrozova Z, Thevenieau F, Le Dall M-T, Hapala I, Papanikolaou S, Chardot T, Nicaud J-M (2008) Control of Lipid Accumulation in the Yeast Yarrowia lipolytica. Appl Environ Microbiol 74:7779–7789PubMedCentralPubMedCrossRefGoogle Scholar
  4. Beopoulos A, Nicaud J-M, Gaillardin C (2011) An overview of lipid metabolism in yeasts and its impact on biotechnological processes. Appl Microbiol Biotechnol 90:1193–1206PubMedCrossRefGoogle Scholar
  5. Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37:911–917PubMedCrossRefGoogle Scholar
  6. Choi JY, Martin CE (1999) The Saccharomyces cerevisiae FAT1 gene encodes an acyl-CoA synthetase that is required for maintenance of very long chain fatty acid levels. J Biol Chem 274:4671–4683PubMedCrossRefGoogle Scholar
  7. Daniëls VW, Smans K, Royaux I, Chypre M, Swinnen JV, Zaidi N (2014) Cancer cells differentially activate and thrive on De Novo lipid synthesis pathways in a low-lipid environment. PLoS ONE 9:e106913PubMedCentralPubMedCrossRefGoogle Scholar
  8. Dey P, Chakraborty M, Kamdar MR, Maiti MK (2014) Functional characterization of two structurally novel diacylglycerol acyltransferase2 isozymes responsible for the enhanced production of stearate-rich storage Lipid in Candida tropicalis SY005. PLoS ONE 9:e94472PubMedCentralPubMedCrossRefGoogle Scholar
  9. Di Paolo G, Kim TW (2011) Linking lipids to Alzheimer’s disease: cholesterol and beyond. Nat Rev Neurosci 12:284–296PubMedCentralPubMedCrossRefGoogle Scholar
  10. Folch J, Lees M, Sloane Stanley GH (1957) A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem 226:497–509PubMedGoogle Scholar
  11. Greenspan P, Fowler SD (1985) Spectrofluorometric studies of the lipid probe, nile red. J Lipid Res 26:781–789PubMedGoogle Scholar
  12. Grimard V, Massier J, Richter D, Schwudke D, Kalaidzidis Y, Fava E, Hermetter A, Thiele C (2008) siRNA screening reveals JNK2 as an evolutionary conserved regulator of triglyceride homeostasis. J Lipid Res 49:2427–2440PubMedCrossRefGoogle Scholar
  13. Hutchins PM, Barkley RM, Murphy RC (2008) Separation of cellular nonpolar neutral lipids by normal-phase chromatography and analysis by electrospray ionization mass spectrometry. J Lipid Res 49:804–813PubMedCentralPubMedCrossRefGoogle Scholar
  14. Kalscheuer R, Luftmann H, Steinbuchel A (2004) Synthesis of novel lipids in Saccharomyces cerevisiae by heterologous expression of an unspecific bacterial acyltransferase. Appl Environ Microbiol 70:7119–7125PubMedCentralPubMedCrossRefGoogle Scholar
  15. Kimura K, Yamaoka M, Kamisaka Y (2004) Rapid estimation of lipids in oleaginous fungi and yeasts using Nile red fluorescence. J Microbiol Methods 56:331–338PubMedCrossRefGoogle Scholar
  16. Kohlwein SD, Veenhuis M, van der Klei IJ (2013) Lipid droplets and peroxisomes: key players in cellular lipid homeostasis or a matter of fat–store ‘em up or burn ‘em down. Genetics 193:1–50PubMedCentralPubMedCrossRefGoogle Scholar
  17. Long AP, Manneschmidt AK, VerBrugge B, Dortch MR, Minkin SC, Prater KE, Biggerstaff JP, Dunlap JR, Dalhaimer P (2012) Lipid droplet de novo formation and fission are linked to the cell cycle in fission yeast. Traffic 13:705–714 Google Scholar
  18. Oelkers P, Tinkelenberg A, Erdeniz N, Cromley D, Billheimer JT, Sturley SL (2000) A lecithin cholesterol acyltransferase-like gene mediates diacylglycerol esterification in yeast. J Biol Chem 275:15609–15612PubMedCrossRefGoogle Scholar
  19. Oelkers P, Cromley D, Padamsee M, Billheimer JT, Sturley SL (2002) The DGA1 gene determines a second triglyceride synthetic pathway in yeast. J Biol Chem 277:8877–8881PubMedCrossRefGoogle Scholar
  20. Oresic M et al (2008) Dysregulation of lipid and amino acid metabolism precedes islet autoimmunity in children who later progress to type 1 diabetes. J Exp Med 205:2975–2984PubMedCentralPubMedCrossRefGoogle Scholar
  21. Petranovic D, Tyo K, Vemuri GN, Nielsen J (2010) Prospects of yeast systems biology for human health: integrating lipid, protein and energy metabolism. FEMS Yeast Res 10:1046–1059PubMedCrossRefGoogle Scholar
  22. Poli JS, Lutzhoft HC, Karakashev DB, Valente P, Angelidaki I (2014) An environmentally-friendly fluorescent method for quantification of lipid contents in yeast. Bioresour Technol 151:388–391PubMedCrossRefGoogle Scholar
  23. Runguphan W, Keasling JD (2014) Metabolic engineering of Saccharomyces cerevisiae for production of fatty acid-derived biofuels and chemicals. Metab Eng 21:103–113PubMedCrossRefGoogle Scholar
  24. Sandager L, Gustavsson MH, Ståhl U, Dahlqvist A, Wiberg E, Banas A, Lenman M, Ronne H, Stymne S (2002) Storage lipid synthesis is non-essential in yeast. J Biol Chem 277:6478–6482PubMedCrossRefGoogle Scholar
  25. Santamauro F, Whiffin FM, Scott RJ, Chuck CJ (2014) Low-cost lipid production by an oleaginous yeast cultured in non-sterile conditions using model waste resources. Biotechnol Biofuels 7:34PubMedCentralPubMedCrossRefGoogle Scholar
  26. Shahidi F (2001) Extraction and Measurement of Total Lipids. In: Decker E (ed) Current protocols in food analytical chemistry. Wiley, New YorkGoogle Scholar
  27. Sitepu IR, Ignatia L, Franz AK, Wong DM, Faulina SA, Tsui M, Kanti A, Boundy-Mills K (2012) An improved high-throughput Nile red fluorescence assay for estimating intracellular lipids in a variety of yeast species. J Microbiol Methods 91:321–328PubMedCentralPubMedCrossRefGoogle Scholar
  28. Sorger D, Athenstaedt K, Hrastnik C, Daum G (2004) A yeast strain lacking lipid particles bears a defect in ergosterol formation. J Biol Chem 279:31190–31196PubMedCrossRefGoogle Scholar
  29. Tai M, Stephanopoulos G (2013) Engineering the push and pull of lipid biosynthesis in oleaginous yeast Yarrowia lipolytica for biofuel production. Metab Eng 15:1–9PubMedCrossRefGoogle Scholar
  30. Wachtler V, Rajagopalan S, Balasubramanian MK (2003) Sterol rich plasma membrane domains in the fission yeast Schizosaccharomyces pombe. J Cell Sci 116:867–874Google Scholar
  31. Watkins PA, Lu JF, Steinberg SJ, Gould SJ, Smith KD, Braiterman LT (1998) Disruption of the Saccharomyces cerevisiae FAT1 gene decreases very long-chain fatty acyl-CoA synthetase activity and elevates intracellular very long-chain fatty acid concentrations. J Biol Chem 273:18210–18219PubMedCrossRefGoogle Scholar
  32. Yazawa H, Kumagai H, Uemura H (2012) Characterization of triglyceride lipase genes of fission yeast Schizosaccharomyces pombe. Appl Microbiol Biotechnol 96:981–991PubMedCrossRefGoogle Scholar
  33. Zhang F, Du G (2012) Dysregulated lipid metabolism in cancer. World J Biol Chem 3:167–174PubMedCentralPubMedCrossRefGoogle Scholar
  34. Zhang Q, Chieu HK, Low CP, Zhang S, Heng CK, Yang H (2003) Schizosaccharomyces pombe cells deficient in triacylglycerols synthesis undergo apoptosis upon entry into the stationary phase. J Biol Chem 278:47145–47155PubMedCrossRefGoogle Scholar

Copyright information

© Springer International Publishing Switzerland 2015

Authors and Affiliations

  • Kerry A. Rostron
    • 1
  • Carole E. Rolph
    • 1
  • Clare L. Lawrence
    • 1
  1. 1.School of Pharmacy and Biomedical SciencesUniversity of Central LancashirePrestonUK

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