The longevity of tor1Δ, sch9Δ, and ras2Δ mutants depends on actin dynamics in Saccharomyces cerevisiae
© Liu et al.; licensee BioMed Central. 2015
Received: 23 November 2014
Accepted: 31 March 2015
Published: 18 April 2015
Recent studies have revealed the role of actin dynamics in the regulation of yeast aging. Although the target of rapamycin (TOR) complex, serine/threonine kinase Sch9, and Ras2 have been shown to play important roles in aging for a long time, the relationship between these regulators and actin has not yet been reported. In this study we investigated the roles of actin polarization in tor1Δ, sch9Δ, and ras2Δ mutant cells. We found that the actin structures in tor1Δ, sch9Δ, and ras2Δ mutant cells were more dynamic than those in the wild type. Destruction of the actin structures with jasplakinolide decreased the life span of tor1Δ, sch9Δ, and ras2Δ mutants. Furthermore, deletion of SLA1 in tor1Δ, sch9Δ, and ras2Δ mutants inhibited the actin dynamics and life span. In addition, we found that the actin cytoskeleton of the long-lived mutant sch9Δ, depended on the transcription factors RIM15 and MSN2/4, but not GIS1, while the actin skeleton of the tor1Δ and ras2Δ mutants depended on RIM15 as expected. Our data suggest that the longevity of tor1Δ, sch9Δ, and ras2Δ mutants is dependent on actin dynamics.
KeywordsTor1 Sch9 Ras2 Longevity Actin dynamic
The budding yeast, Saccharomyces cerevisiae, has been successfully established as a model to study the mechanisms of aging. Currently, there are two models to study aging in yeast: the replicative life span (RLS), which represents the replicative capacity of a mother cell and is measured by counting the number of daughter cells produced by a mother cell [1,2], and the chronological life span (CLS), which is defined as the length of time the mother cell can survive in liquid medium [3,2].
Tor1 is a PIK-related protein kinase and one of the subunits of TOR complex 1 (TORC1). The TOR complex controls growth in response to nutrients by regulating translation, transcription, ribosome biogenesis, nutrient transport, and autophagy, which are important cellular responses implicated in increased life span . Previous studies suggest that the TOR pathway controls mitochondrial respiration and production of reactive oxygen species (ROS) in yeast, which is thought to be an important determinant for life span in yeast cells [5-7].
The yeast kinase Sch9 can mediate glucose-dependent signaling, stimulate growth and cellular proliferation, and decrease stress resistance [8,9]. The COOH-terminal region of Sch9 is highly homologous to the AGC family members of serine/threonine kinases, which include Akt/PKB and S6K in mammalian cells. Sch9 can be phosphorylated directly by TOR1 at multiple sites and by PDK1 in the activation loop . Major scientific discoveries into the regulation of longevity by Sch9 in yeast comes from Longo’s Lab . They have shown that sch9Δ cells can survive three times longer than wild type cells. Stress-resistance transcription factors, such as Msn2/4 and protein kinase, Rim15, are required for this life span extension .
RAS2 is a homolog of the mammalian oncogene, RAS, and is highly related to the RAS1 gene in budding yeast . It codes for a small GTP-binding protein and has been shown to regulate the nitrogen starvation response through its effects on adenylate cyclase (encoded by the CYR1 gene). The GTP-bound form, Ras2, directly induces the production of cAMP by adenylate cyclase .
Previous studies have shown that actin dynamics regulates longevity though its underlying mechanism is still unclear [13,14]. It was suggested that decreased actin dynamics led to depolarization of the mitochondria and induced production of reactive oxygen species (ROS), which reduced cell longevity. Tor1, Sch9 and Ras2 are important regulators of aging, however, whether actin dynamics is required for longevity of tor1Δ, sch9Δ, and ras2Δ mutants is unknown.
In this study, the role of actin dynamics in the life span of tor1Δ, sch9Δ, and ras2Δ mutant yeast cells was investigated. Using genetic and biochemistry assays, we found that the actin pattern is more dynamic in tor1Δ, sch9Δ, and ras2Δ mutant cells than that in wild type cells, and that reduced actin dynamics, through either the actin stabilizing drug, jasplakinolide, or SLA1 deletion, led to shorter life spans in these mutants. Although actin dynamics is required for all three mutants, the actin cytoskeleton in the long-lived mutant sch9Δ, depended on the transcription factors, RIM15 and MSN2/4, while tor1Δ and ras2Δ depended on RIM15. These data suggest that the regulation of actin dynamics in tor1Δ, sch9Δ, and ras2Δ is based on different mechanisms.
The actin structure of tor1Δ, sch9Δ, and ras2Δ mutants is more dynamic than that of wild type cells
In order to test if the tor1Δ, sch9Δ, and ras2Δ strains used in this study have similar extended life spans as in the previous study, the same medium was used to examine the chronological life span as noted in the previous study . As expected, tor1Δ, sch9Δ, and ras2Δ was smaller in size than the DBY746 wild type cells (data not shown). At day 8 after inoculation, the viability of DBY746 was less than 5%. The viability of tor1Δ, sch9Δ, and ras2Δ cells at the same time points, however, was 73%, 85%, and 82%, respectively. At day 11, almost all the wild type cells were dead, but the viability of tor1Δ, sch9Δ, and ras2Δ cells was 38%,78%, and 73%, respectively. These data suggest that the yeast mutants tor1Δ, sch9Δ, and ras2Δ have longer life spans, as reported previously .
Jasplakinolide treatment decreases the actin dynamics and life span of tor1Δ, sch9Δ, and ras2Δ mutants
The deletion of SLA1 in tor1Δ, sch9Δ, and ras2Δ mutants inhibits actin dynamics and life span
The actin cytoskeleton dynamics in sch9Δ mutant cells depends on Rim15 and Msn2/4, while it relies on Rim15 in tor1∆ and ras2∆ mutants
A recent study showed that Tor1, Sch9, and Ras2 can regulate carbon source substitution and control calorie restriction in life span extension . Although actin has long been proposed to play an important role in aging , whether actin dynamics is involved in the increased life span of tor1∆, sch9Δ, and ras2∆ mutants has never been examined. To answer this question, we examined actin organization in the wild type, tor1∆, sch9Δ, and ras2∆ mutants by fluorescence microscopy. Consistent with previous reports, the actin structure is dynamic in these longevity mutants but not wild type cells. Numerous bright small separate dots around the cell were found in tor1∆, sch9Δ, and ras2∆ mutant cells. To the contrary, actin in the DBY746 wild type looks like a large aggregate clump after day 3 or 5 cultures. The aggregates were shown to be extremely stable structures and less dynamic , but the separate smaller dots were not. To further confirm the role of actin dynamics in the aging process, the actin structure was destroyed either by jasplakinolide treatment or through deletion of the SLA1 gene. Our data indicate that actin dynamics is required for the increased life span of the sch9Δ, tor1∆, and ras2∆ mutants. Because RIM15, GIS1, and MSN2/4 are effectors of Tor1, Sch9, or Ras2, which are also shown to regulate life span, we speculate that some of them may be required for maintenance of the dynamic actin pattern in tor1∆, sch9Δ, and ras2∆ mutants. The fact that the actin cytoskeleton dynamics in sch9Δ depends on RIM15 and MSN2/4 but not GIS1, and in tor1∆ and ras2∆, depends on RIM15, suggests that these effectors also control actin dynamics in sch9Δ, tor1∆ and ras2∆ mutants. Our study suggested that dynamic actin pattern is required for longevity of sch9Δ, tor1∆ and ras2∆ mutants. These results will help us to further understand the role of actin in aging.
The mechanism that these mutants regulate actin dynamics, however, is still unclear. In budding yeast, Tor1 functions upstream of the Rho1-Pkc1 pathway, which was shown to regulates actin dynamics . Ho and Bretscher demonstrated that deletion of RAS2 results in loss of Cdc42p and F-actin polarity , and it was suggested that Ras2 regulates actin cytoskeleton dynamics via the actin regulatory factor Srv2 . Although there is no direct evidence shows that Sch9 regulates actin dynamics, recent study indicated that Sch9 is a key regulator of sphingolipid metabolism , which was shown to be able to regulate actin cytoskeleton through Ypk1 and Pkc1 . These data suggest that Sch9 may indirectly controls actin dynamics. In addition, the effectors of Rim15, Gis1, and Msn2/4 may also mediate actin dynamic pattern during aging. Further investigation is required to answer these questions.
Yeast strains and media
Yeast strains used in this study
MATα leu2-3 112 his3∆1 trp1-289 ura3-52 GAL+
MATα leu2-3 112 his3∆1 trp1-289 ura3-52 tor1::KanMX GAL+
MATα leu2-3 112 his3∆1 trp1-289 ura3-52 sch9::KanMX GAL+
MATα leu2-3 112 his3∆1 trp1-289 ura3-52 ras2::KanMX GAL+
MATα leu2-3 112 his3∆1 trp1-289 ura3-52 tor1::KanMX rim15::TRP1 GAL+
MATα leu2-3 112 his3∆1 trp1-289 ura3-52 sch9::KanMX rim15::TRP1 GAL+
MATα leu2-3 112 his3∆1 trp1-289 ura3-52 sch9::KanMX gis1::HIS3 GAL+
MATα leu2-3 112 his3∆1 trp1-289 ura3-52 sch9::KanMX msn2::HIS3 msn4::LEU2 GAL+
MATα leu2-3 112 his3∆1 trp1-289 ura3-52 ras2::KanMX rim15::TRP1 GAL+
MATα leu2-3 112 his3∆1 trp1-289 ura3-52 ras2::KanMX gis1::HIS3 GAL+
MATα leu2-3 112 his3∆1 trp1-289 ura3-52 tor1::KanMX sla1::TRP1 GAL+
MATα leu2-3 112 his3∆1 trp1-289 ura3-52 sch9::KanMX sla1::TRP1 GAL+
MATα leu2-3 112 his3∆1 trp1-289 ura3-52 ras2::KanMX sla1::TRP1 GAL+
To avoid possible effects due to auxotrophic deficiencies of the strains, the liquid synthetic complete dextrose (SCD) medium was supplemented with a 4-fold excess of tryptophan, leucine, uracil, and histidine (17) and 2% dextrose was used as a carbon source. This, we called “aging medium.” For YPD plates, 19 g L−1 bacto-yeast extract, 20 g L−1 bacto-peptone, and 20 g L−1 bacto-agar (Becton, Dickenson and Company) were dissolved in water and autoclaved and dextrose was added to a final concentration of 2%. YPE and YPG plates were made in the same way as YPD plates, except that 3% ethanol or 3% glycerol was used as a carbon source instead of dextrose.
Chronological life span assay
The chronological life span was measured according to the previous report . Briefly, a small amount of yeast strain from the frozen (−80°C) sample was streaked onto an YPD plate and incubated at 30°C for 2–3 days. Five to six colonies were inoculated into 2 ml YPD liquid medium to make the saturated overnight culture (SONC). The fresh SONC was diluted in 25 ml of liquid medium to an initial density of ~1 × 106 cells mL−1 (A600 = 0.1). The flasks were maintained at 30°C at 220 rpm in the air bath shaker. The initial time point began at day 0, the next day was day 1, and so on. At day 3, the cells reached the stationary phase and stopped dividing, at which point the cells were deemed to be 100% viable. To determine the number of viable cells, 100-μl aliquots were removed from each culture and diluted serially to reach a 1:105 dilution in sterile distilled water. Two dilutions per culture were used routinely, and 100 μl of the dilution was plated onto YPD plates in duplicate. After incubation for 2–3 days, the number of colonies was counted, and the viability at this time point was calculated by comparing the numbers to day 0.
Phalloidin-TRITC (Sigma, P1951) stain was performed as previously reported . Cells were fixed for 1 h by adding 37% formaldehyde solution directly to growth medium to a final concentration of 3.7%. After washing with 1× PBS solution three times, cells were stained with phalloidin-TRITC in the dark at room temperature for 1 h. A Nikon Eclipse E600 fluorescence microscope equipped with a Plan Apo 100× 1.4 oil immersion objective was used for fluorescence microscopy. The images were captured with a SPOT RT 9.0 onochrome-6 camera using the MetaMorph (version 6.3.0) acquisition software. The whole cell reconstruction was gained by confocal microscopy (Leica TSC SP2/AOBS). The thickness of the Z-sections is 400 nm. Images were processed by Adobe Photoshop (version 7.0).
This work was supported by the National Natural Science Foundation of China (No. 81173123) and Liaoning Natural Science Foundation (NO.2013225049). We are very grateful to Dr. Robert C. Dickson (University of Kentucky), who gave the author’s post-doc training in the aging-related academic field and his generous stains, plasmids and technical assistance.
- Mortimer RK, Johnston JR. Life span of individual yeast cells. Nature. 1959;183:1751–2.View ArticlePubMedGoogle Scholar
- Lavoie H, Whiteway M. Increased respiration in the sch9 Delta mutant is required for increasing chronological life span but not replicative life span. Eukaryot Cell. 2008;7:1127–35.View ArticlePubMed CentralPubMedGoogle Scholar
- Fabrizio P, Longo VD. The chronological life span of Saccharomyces cerevisiae. Aging Cell. 2003;2:73–81.View ArticlePubMedGoogle Scholar
- Cornu M, Albert V, Hall MN. mTOR in aging, metabolism, and cancer. Curr Opin Genet Dev. 2013;23:53–62.View ArticlePubMedGoogle Scholar
- Bonawitz ND, Chatenay-Lapointe M, Pan Y, Shadel GS. Reduced TOR signaling extends chronological life span via increased respiration and upregulation of mitochondrial gene expression. Cell Metab. 2007;5:265–77.View ArticlePubMed CentralPubMedGoogle Scholar
- Pan Y, Shadel GS. Extension of chronological life span by reduced TOR signaling requires down-regulation of Sch9p and involves increased mitochondrial OXPHOS complex density. Aging. 2009;1:131–45.PubMed CentralPubMedGoogle Scholar
- Schieke SM, Finkel T. TOR and aging: less is more. Cell Metab. 2007;5:233–5.View ArticlePubMedGoogle Scholar
- Kandel ES, Hay N. The regulation and activities of the multifunctional serine/threonine kinase Akt/PKB. Exp Cell Res. 1999;253:210–29.View ArticlePubMedGoogle Scholar
- Thevelein JM, de Winde JH. Novel sensing mechanisms and targets for the cAMP-protein kinase A pathway in the yeast Saccharomyces cerevisiae. Mol Microbiol. 1999;33:904–18.View ArticlePubMedGoogle Scholar
- Urban J, Soulard A, Huber A, Lippman S, Mukhopadhyay D, Deloche O, et al. Sch9 is a major target of TORC1 in Saccharomyces cerevisiae. Mol Cell. 2007;26:663–74.View ArticlePubMedGoogle Scholar
- Fabrizio P, Pozza F, Pletcher SD, Gendron CM, Longo VD. Regulation of longevity and stress resistance by Sch9 in yeast. Science. 2001;292:288–90.View ArticlePubMedGoogle Scholar
- Wei M, Fabrizio P, Hu J, Ge HY, Cheng C, Li L, et al. Life span extension by calorie restriction depends on Rim15 and transcription factors downstream of Ras/PKA, Tor, and Sch9. PLoS Genet. 2008;4:139–49.View ArticleGoogle Scholar
- Gourlay CW, Carpp LN, Timpson P, Winder SJ, Ayscough KR. A role for the actin cytoskeleton in cell death and aging in yeast. J Cell Biol. 2004;164:803–9.View ArticlePubMed CentralPubMedGoogle Scholar
- Gourlay CW, Ayscough KR. A role for actin in aging and apoptosis. Biochem Soc Trans. 2005;33:1260–4.View ArticlePubMedGoogle Scholar
- Warren DT, Andrews PD, Gourlay CW, Ayscough KR. Sla1p couples the yeast endocytic machinery to proteins regulating actin dynamics. J Cell Sci. 2002;115:1703–15.PubMedGoogle Scholar
- Ayscough KR. Endocytosis and the development of cell polarity in yeast require a dynamic F-actin cytoskeleton. Curr Biol. 2000;10:1587–90.View ArticlePubMedGoogle Scholar
- Wei M, Fabrizio P, Madia F, Hu J, Ge HY, Li LM, et al. Tor1/Sch9-regulated carbon source substitution is as effective as calorie restriction in life span extension. PLoS Genet. 2009;5, e1000467.View ArticlePubMed CentralPubMedGoogle Scholar
- Torres J, Di Como CJ, Herrero E, De La Torre-Ruiz MA. Regulation of the cell integrity pathway by rapamycin-sensitive TOR function in budding yeast. J Biol Chem. 2002;277:43495–504.View ArticlePubMedGoogle Scholar
- Ho J, Bretscher A. Ras regulates the polarity of the yeast actin cytoskeleton through the stress response pathway. Mol Biol Cell. 2001;12:1541–55.View ArticlePubMed CentralPubMedGoogle Scholar
- Gourlay CW, Ayscough KR. Actin-induced hyperactivation of the Ras signaling pathway leads to apoptosis in Saccharomyces cerevisiae. Mol Cell Biol. 2006;26:6487–501.View ArticlePubMed CentralPubMedGoogle Scholar
- Swinnen E, Wilms T, Idkowiak-Baldys J, Smets B, De Snijder P, Accardo S, et al. The protein kinase Sch9 is a key regulator of sphingolipid metabolism in Saccharomyces cerevisiae. Mol Biol Cell. 2014;25:196–211.View ArticlePubMed CentralPubMedGoogle Scholar
- Dickson RC. Roles for sphingolipids in Saccharomyces cerevisiae. Adv Exp Med Biol. 2010;688:217–31.View ArticlePubMedGoogle Scholar
- Sherman F. Getting started with yeast. Methods Enzymol. 2002;350:3–41.View ArticlePubMedGoogle Scholar
- Longtine MS, McKenzie 3rd A, Demarini DJ, Shah NG, Wach A, Brachat A, et al. Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast. 1998;14:953–61.View ArticlePubMedGoogle Scholar
- Parrella E, Longo VD. The chronological life span of Saccharomyces cerevisiae to study mitochondrial dysfunction and disease. Methods. 2008;46:256–62.View ArticlePubMedGoogle Scholar
- Madeo F, Frohlich E, Ligr M, Grey M, Sigrist SJ, Wolf DH, et al. Oxygen stress: a regulator of apoptosis in yeast. J Cell Biol. 1999;145:757–67.View ArticlePubMed CentralPubMedGoogle Scholar
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