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Pizza club - March 2017 - Ursula

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A. Hughes, DE Gottschling, An early age increase in vacuolar pH limits mitochondrial function and lifespan in yeast, Nature 492, 261–265 (13 December 2012), doi:10.1038/nature11654

Adam L. Hughes & Daniel E. Gottschling

Published in: Science
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Pizza club - March 2017 - Ursula

  1. 1. 1 Ursula Heins Marroquín Journal Club 22.03.17
  2. 2. 2 Saccharomyces cerevisiae (Budding yeast) unicellular eukaryotic organism small genome comprising about 6000 genes, which has been completely sequenced molecular genetic tools Cost effective model Easy to manipulate High throughput screening 31% of yeast gene has a human homolog
  3. 3. 3 Introduction S. cerevisiae Aged yeast cells - Mitochondria fragmentation - Increase ROS - Loss of mitochondrial DNA AGEING Chronological life span Replicative life span S. cerevisiae
  4. 4. 4 Introduction S. cerevisiae Aged yeast cells - Mitochondria fragmentation - Increase ROS - Loss of mitochondrial DNA AGEING Chronological life span Replicative life span S. cerevisiae Number of times an individual cell divides
  5. 5. 5 Introduction: Classical RLS
  6. 6. 6 Method: Mother enrichment program (MEP) Mother Cell Cell division Daughter Cell
  7. 7. 7 Method: Mother enrichment program (MEP) Daughter Cell the transcription factor Ace2 Sequester in the cytoplasm Transported into the nucleus
  8. 8. 8 Method: Mother enrichment program (MEP) Cre- EBD78 SUMO-conjugating enzyme Activator of the anaphase-promoting complex (APC) Arrest in M-phase
  9. 9. 9 Results: Mitochondrial dysfunction in aged yeast Calcofluor staining of the bud scar TOM70-GFP Mitochondrial marker Nr. Cell division Characterization of age-associated mitochondrial phenotypes
  10. 10. 10 Results: Decreased of mitochondrial function DiOC6 : ΔΨ-dependent mitochondrial fluorescent dye
  11. 11. 11 Mitochondrial structure in young cells 250 genes1) 2) Overexpression of the 250 genes MEP strain expressing Tom70-GFP Delayed to the onset of altered mitochondrial morphology in aged cells
  12. 12. 12 Results: VAM1 and VPH2 VAM1 VPH2 A Catalytic subunit of V-ATPase V-ATPase assembly V-ATPase : Acidification of the vacuole Overexpression of VMA suppressed mitochondrial dysfunction in 93 % Overexpression of VPH2 suppressed mitochondrial dysfunction in 77 % Suppression by VMA1 or VPH2 overexpression required V-ATPase activity
  13. 13. 13 Results: Vacuolar and mitochondrial function are linked • Overexpression of VMA1 and VPH2 increase both media and maximal replicative lifespan.
  14. 14. 14 How vpH affects mitochondrial function? vacuolar protein degradation? Deletion of PEP4 No alteration of mitochondrial structure
  15. 15. 15 How vpH affects mitochondrial function? Import of vacuolar metabolite? Overexpression of vacuolar importers + ConcA ATV1 Neutral amino acid transporter Prevent mitochondrial dysfunction
  16. 16. 16 Results: ATV1 prevents mitochondrial fragmentation Overexpression of ATV1 • Prevent alteration of mitochondrial structure • No change in vacuolar acidification
  17. 17. 17 Results: ATV1 as aging modulator Deletion of ATV1 Development of age-induced mitochondrial dysfunction Overexpression = Extending RLS Deletion = Shortening of RLS
  18. 18. 18 Conclusion o Yeast is a good model for aging o Link between vacuolar pH and mitochondrial function o Link between metabolite transport and aging o Vacuole functionalion as aging modulator o Reestablishment of acidification in the vacuole in the daughter cells
  19. 19. 19 Thank you

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