A Rapid, High-throughput Method for Determining Chronological Lifespan in Budding Yeast

Overview

Journal J Biol Methods

Specialty Biology

Date 2019 Aug 28

PMID 31453256

Citations 2

Authors

Zachery R Belak

Troy Harkness

Christopher H Eskiw

Affiliations

Soon will be listed here.

Abstract

The budding yeast 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.

Citing Articles

Barcode sequencing and a high-throughput assay for chronological lifespan uncover ageing-associated genes in fission yeast.

Romila C, Townsend S, Malecki M, Kamrad S, Rodriguez-Lopez M, Hillson O Microb Cell. 2021; 8(7):146-160.

PMID: 34250083 PMC: 8246024. DOI: 10.15698/mic2021.07.754.

Genome-wide screens in yeast models towards understanding chronological lifespan regulation.

Legon L, Rallis C Brief Funct Genomics. 2021; 21(1):4-12.

PMID: 33728458 PMC: 8834652. DOI: 10.1093/bfgp/elab011.

References

Defossez P, Prusty R, Kaeberlein M, Lin S, Ferrigno P, Silver P . Elimination of replication block protein Fob1 extends the life span of yeast mother cells. Mol Cell. 1999; 3(4):447-55. DOI: 10.1016/s1097-2765(00)80472-4. View

Fabrizio P, Pozza F, Pletcher S, Gendron C, Longo V . Regulation of longevity and stress resistance by Sch9 in yeast. Science. 2001; 292(5515):288-90. DOI: 10.1126/science.1059497. View

Harkness T, Davies G, Ramaswamy V, Arnason T . The ubiquitin-dependent targeting pathway in Saccharomyces cerevisiae plays a critical role in multiple chromatin assembly regulatory steps. Genetics. 2002; 162(2):615-32. PMC: 1462303. DOI: 10.1093/genetics/162.2.615. View

Howitz K, Bitterman K, Cohen H, Lamming D, Lavu S, Wood J . Small molecule activators of sirtuins extend Saccharomyces cerevisiae lifespan. Nature. 2003; 425(6954):191-6. DOI: 10.1038/nature01960. View

Harkness T, Shea K, Legrand C, Brahmania M, Davies G . A functional analysis reveals dependence on the anaphase-promoting complex for prolonged life span in yeast. Genetics. 2004; 168(2):759-74. PMC: 1448841. DOI: 10.1534/genetics.104.027771. View

Harkness T, Arnason T, Legrand C, Pisclevich M, Davies G, Turner E . Contribution of CAF-I to anaphase-promoting-complex-mediated mitotic chromatin assembly in Saccharomyces cerevisiae. Eukaryot Cell. 2005; 4(4):673-84. PMC: 1087812. DOI: 10.1128/EC.4.4.673-684.2005. View

Berridge M, Herst P, Tan A . Tetrazolium dyes as tools in cell biology: new insights into their cellular reduction. Biotechnol Annu Rev. 2005; 11:127-52. DOI: 10.1016/S1387-2656(05)11004-7. View

Powers 3rd R, Kaeberlein M, Caldwell S, Kennedy B, Fields S . Extension of chronological life span in yeast by decreased TOR pathway signaling. Genes Dev. 2006; 20(2):174-84. PMC: 1356109. DOI: 10.1101/gad.1381406. View

Medvedik O, Sinclair D . Caloric restriction and life span determination of yeast cells. Methods Mol Biol. 2007; 371:97-109. DOI: 10.1007/978-1-59745-361-5_9. View

10.

Medvedik O, Lamming D, Kim K, Sinclair D . MSN2 and MSN4 link calorie restriction and TOR to sirtuin-mediated lifespan extension in Saccharomyces cerevisiae. PLoS Biol. 2007; 5(10):e261. PMC: 1994990. DOI: 10.1371/journal.pbio.0050261. View

11.

Evans D, Kapahi P, Hsueh W, Kockel L . TOR signaling never gets old: aging, longevity and TORC1 activity. Ageing Res Rev. 2010; 10(2):225-37. PMC: 2943975. DOI: 10.1016/j.arr.2010.04.001. View

12.

Sanchez N, Konigsberg M . Using yeast to easily determine mitochondrial functionality with 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenyltetrazolium bromide (MTT) assay. Biochem Mol Biol Educ. 2011; 34(3):209-12. DOI: 10.1002/bmb.2006.49403403209. View

13.

Geissmann Q . OpenCFU, a new free and open-source software to count cell colonies and other circular objects. PLoS One. 2013; 8(2):e54072. PMC: 3574151. DOI: 10.1371/journal.pone.0054072. View

14.

Menzel J, Malo M, Chan C, Prusinkiewicz M, Arnason T, Harkness T . The anaphase promoting complex regulates yeast lifespan and rDNA stability by targeting Fob1 for degradation. Genetics. 2013; 196(3):693-709. PMC: 3948801. DOI: 10.1534/genetics.113.158949. View

15.

Postnikoff S, Harkness T . Replicative and chronological life-span assays. Methods Mol Biol. 2014; 1163:223-7. DOI: 10.1007/978-1-4939-0799-1_17. View

16.

Kwolek-Mirek M, Zadrag-Tecza R . Comparison of methods used for assessing the viability and vitality of yeast cells. FEMS Yeast Res. 2014; 14(7):1068-79. DOI: 10.1111/1567-1364.12202. View

17.

Malo M, Postnikoff S, Arnason T, Harkness T . Mitotic degradation of yeast Fkh1 by the Anaphase Promoting Complex is required for normal longevity, genomic stability and stress resistance. Aging (Albany NY). 2016; 8(4):810-30. PMC: 4925830. DOI: 10.18632/aging.100949. View

18.

Gillespie Z, Pickering J, Eskiw C . Better Living through Chemistry: Caloric Restriction (CR) and CR Mimetics Alter Genome Function to Promote Increased Health and Lifespan. Front Genet. 2016; 7:142. PMC: 4988992. DOI: 10.3389/fgene.2016.00142. View

19.

Maslanka R, Kwolek-Mirek M, Zadrag-Tecza R . Consequences of calorie restriction and calorie excess for the physiological parameters of the yeast Saccharomyces cerevisiae cells. FEMS Yeast Res. 2017; 17(8). DOI: 10.1093/femsyr/fox087. View

20.

Tyler J, Johnson J . The role of autophagy in the regulation of yeast life span. Ann N Y Acad Sci. 2018; 1418(1):31-43. PMC: 5934334. DOI: 10.1111/nyas.13549. View