A rapid, high-throughput method for determining chronological lifespan in budding yeast
The budding yeast Saccharomyces cerevisiae is a major model system in the study of aging. Like metazoans, yeast lifespan is extended by caloric restriction and treatment with pharmacological agents which extend lifespan. A major workhorse of aging research in budding yeast is the chronological lifespan assay. Traditionally, chronological lifespan assays consist of taking regular samples of aging yeast cultures, plating out aliquots on agar, and counting the resulting colonies. This method, while highly reliable, is labor-intensive and expensive in terms of materials consumed. Here, we report a novel MTT-based method for assessing chronological lifespan in yeast. We show that this method is equal to the colony counting method in its rigorous and reliable measurement of lifespan extension in yeast as a result of caloric restriction, and is able to distinguish known long-lived and short-lived yeast strains. We have further developed this method into a high-throughput assay that allows rapid screening of potential anti-aging compounds as well as yeast strains with altered lifespan. Application of this method permits the rapid identification of anti-aging activities in yeast and may facilitate identification of materials with therapeutic potential for higher animals and, most importantly, humans.
1. Postnikoff SD, Harkness TA (2014) Replicative and chronological life-span assays. Methods Mol Biol 1163: 223-227.
2. Harkness TAA (2018) Activating the Anaphase Promoting Complex to Enhance Genomic Stability and Prolong Lifespan. Int J Mol Sci 19.
3. Baccolo G, Stamerra G, Damiano Pellegrino C, Orlandi I, Vai M (2018) Mitochondrial Metabolism and Aging in Yeast. Int Rev Cell Mol Biol 340: 1-33.
4. Tyler JK, Johnson JE (2018) The role of autophagy in the regulation of yeast life span. Ann N Y Acad Sci 1418: 31-43.
5. Mohammad K, Titorenko VI (2018) Yeast chronological aging is linked to cell cycle regulation. Cell Cycle: 1-2.
6. Gillespie ZE, Pickering J, Eskiw CH (2016) Better Living through Chemistry: Caloric Restriction (CR) and CR Mimetics Alter Genome Function to Promote Increased Health and Lifespan. Front Genet 7: 142.
7. Markaki MM, A.; Tavernarakis, N. (2018) The Role of Autophagy in Aging: Molecular Mechanisms. In: Hayat MA, editor. Autophagy: Cancer, Other Pathologies, Inflammation, Immunity, Infection, and Aging: Academic Press. pp. 123-138.
8. Medvedik O, Sinclair DA (2007) Caloric restriction and life span determination of yeast cells. Methods Mol Biol 371: 97-109.
9. Arlia-Ciommo A, Leonov A, Beach A, Richard VR, Bourque SD, et al. (2018) Caloric restriction delays yeast chronological aging by remodeling carbohydrate and lipid metabolism, altering peroxisomal and mitochondrial functionalities, and postponing the onsets of apoptotic and liponecrotic modes of regulated cell death. Oncotarget 9: 16163-16184.
10. Maslanka R, Kwolek-Mirek M, Zadrag-Tecza R (2017) Consequences of calorie restriction and calorie excess for the physiological parameters of the yeast Saccharomyces cerevisiae cells. FEMS Yeast Res 17.
11. Zi F, Zi H, Li Y, He J, Shi Q, et al. (2018) Metformin and cancer: An existing drug for cancer prevention and therapy. Oncol Lett 15: 683-690.
12. Evans DS, Kapahi P, Hsueh WC, Kockel L (2011) TOR signaling never gets old: aging, longevity and TORC1 activity. Ageing Res Rev 10: 225-237.
13. Howitz KT, Bitterman KJ, Cohen HY, Lamming DW, Lavu S, et al. (2003) Small molecule activators of sirtuins extend Saccharomyces cerevisiae lifespan. Nature 425: 191-196.
14. Medvedik O, Lamming DW, Kim KD, Sinclair DA (2007) MSN2 and MSN4 link calorie restriction and TOR to sirtuin-mediated lifespan extension in Saccharomyces cerevisiae. PLoS Biol 5: e261.
15. Powers RW, 3rd, Kaeberlein M, Caldwell SD, Kennedy BK, Fields S (2006) Extension of chronological life span in yeast by decreased TOR pathway signaling. Genes Dev 20: 174-184.
16. Kennedy BK, Austriaco NR, Jr., Guarente L (1994) Daughter cells of Saccharomyces cerevisiae from old mothers display a reduced life span. J Cell Biol 127: 1985-1993.
17. Fabrizio P, Pozza F, Pletcher SD, Gendron CM, Longo VD (2001) Regulation of longevity and stress resistance by Sch9 in yeast. Science 292: 288-290.
18. Ediriweera MK, Tennekoon KH, Samarakoon SR (2018) In vitro assays and techniques utilized in anticancer drug discovery. J Appl Toxicol.
19. Berridge MV, Herst PM, Tan AS (2005) Tetrazolium dyes as tools in cell biology: new insights into their cellular reduction. Biotechnol Annu Rev 11: 127-152.
20. Sanchez NS, Konigsberg M (2006) Using yeast to easily determine mitochondrial functionality with 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenyltetrazolium bromide (MTT) assay. Biochem Mol Biol Educ 34: 209-212.
21. Kwolek-Mirek M, Zadrag-Tecza R (2014) Comparison of methods used for assessing the viability and vitality of yeast cells. FEMS Yeast Res 14: 1068-1079.
22. Hodgson VJ, Walker GM, Button D (1994) A rapid colorimetric assay of killer toxin activity in yeast. FEMS Microbiol Lett 120: 201-205.
23. Jahn B, Stuben A, Bhakdi S (1996) Colorimetric susceptibility testing for Aspergillus fumigatus: comparison of menadione-augmented 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide and Alamar blue tests. J Clin Microbiol 34: 2039-2041.
24. Jahn B, Martin E, Stueben A, Bhakdi S (1995) Susceptibility testing of Candida albicans and Aspergillus species by a simple microtiter menadione-augmented 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay. J Clin Microbiol 33: 661-667.
25. Menzel J, Malo ME, Chan C, Prusinkiewicz M, Arnason TG, et al. (2014) The anaphase promoting complex regulates yeast lifespan and rDNA stability by targeting Fob1 for degradation. Genetics 196: 693-709.
26. Harkness TA, Arnason TG, Legrand C, Pisclevich MG, Davies GF, et al. (2005) Contribution of CAF-I to anaphase-promoting-complex-mediated mitotic chromatin assembly in Saccharomyces cerevisiae. Eukaryot Cell 4: 673-684.
27. Harkness TA, Davies GF, Ramaswamy V, Arnason TG (2002) The ubiquitin-dependent targeting pathway in Saccharomyces cerevisiae plays a critical role in multiple chromatin assembly regulatory steps. Genetics 162: 615-632.
28. Malo ME, Postnikoff SD, Arnason TG, Harkness TA (2016) Mitotic degradation of yeast Fkh1 by the Anaphase Promoting Complex is required for normal longevity, genomic stability and stress resistance. Aging (Albany NY) 8: 810-830.
29. Geissmann Q (2013) OpenCFU, a new free and open-source software to count cell colonies and other circular objects. PLoS One 8: e54072.
30. Defossez PA, Prusty R, Kaeberlein M, Lin SJ, Ferrigno P, et al. (1999) Elimination of replication block protein Fob1 extends the life span of yeast mother cells. Mol Cell 3: 447-455.
31. Harkness TA, Shea KA, Legrand C, Brahmania M, Davies GF (2004) A functional analysis reveals dependence on the anaphase-promoting complex for prolonged life span in yeast. Genetics 168: 759-774.