lac-1 and lag-1 with ras-1 affect aging and the biological clock in Neurospora crassa

John K Brunson; James Griffith; Daneisha Bowles; Mary E Case; Jonathan Arnold

doi:10.1002/ece3.2554

lac-1 and lag-1 with ras-1 affect aging and the biological clock in Neurospora crassa

Ecol Evol. 2016 Oct 24;6(23):8341-8351. doi: 10.1002/ece3.2554. eCollection 2016 Dec.

Authors

John K Brunson¹, James Griffith², Daneisha Bowles³, Mary E Case³, Jonathan Arnold³

Affiliations

¹ Center for Marine Biotechnology and Biomedicine J. Craig Venter Institute-West Coast Campus University of California San Diego Scripps Institution of Oceanography La Jolla CA USA.
² College of Agricultural and Environmental Sciences University of Georgia Athens GA USA; Genetics Department University of Georgia Athens GA USA.
³ Genetics Department University of Georgia Athens GA USA.

Abstract

Using an automated cell counting technique developed previously (Case et al., Ecology and Evolution 2014; 4: 3494), we explore the lifespan effects of lac-1, a ceramide synthase gene paralogous to lag-1 in Neurospora crassa in conjunction with the band bd (ras-1) gene. We find that the replicative lifespan of a lac-1^KObd double mutants is short, about one race tube cycle, and this double mutant lacks a strong ~21-hr clock cycle as shown by race tube and fluorometer analysis of fluorescent strains including lac-1^KO . This short replicative lifespan phenotype is contrasted with a very long estimated chronological lifespan for lac-1^KObd double mutants from 247 to 462 days based on our regression analyses on log viability, and for the single mutant lac-1^KO, 161 days. Both of these estimated lifespans are much higher than that of previously studied WT and bd single mutant strains. In a lac-1 rescue and induction experiment, the expression of lac-1⁺ as driven by a quinic acid-dependent promoter actually decreases the median chronological lifespan of cells down to only 7 days, much lower than the 34-day median lifespan found in control bd conidia also grown on quinic acid media, which we interpret as an effect of balancing selection acting on ceramide levels based on previous findings from the literature. Prior work has shown phytoceramides can act as a signal for apoptosis in stressed N. crassa cells. To test this hypothesis of balancing selection on phytoceramide levels, we examine the viability of WT, lag-1^KObd, and lac-1^KObd strains following the dual stresses of heat and glycolysis inhibition, along with phytoceramide treatments of different dosages. We find that the phytoceramide dosage-response curve is altered in the lag-1^KObd mutant, but not in the lac-1^KObd mutant. We conclude that phytoceramide production is responsible for the previously reported longevity effects in the lag-1^KObd mutant, but a different ceramide may be responsible for the longevity effect observed in the lac-1^KObd mutant.

Keywords: Neurospora crassa; aging; balancing selection; biological clock; ceramide; circadian rhythms; lac‐1; lag‐1.