Date post: | 11-Apr-2017 |
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Ursula Heins Marroquín
Journal Club22.03.17
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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
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Introduction
S. cerevisiae
Aged yeast cells- Mitochondria fragmentation- Increase ROS- Loss of mitochondrial DNA
AGEING
Chronological life span
Replicative life spanS. cerevisiae
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Introduction
S. cerevisiae
Aged yeast cells- Mitochondria fragmentation- Increase ROS- Loss of mitochondrial DNA
AGEING
Chronological life span
Replicative life spanS. cerevisiae
Number of times an individual cell divides
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Introduction: Classical RLS
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Method: Mother enrichment program (MEP)
Mother Cell
Cell division
Daughter Cell
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Method: Mother enrichment program (MEP)
Daughter Cell
the transcription factor Ace2
- estr
adiol Sequester in the cytoplasm
+ estradiol Transported into the nucleus
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Method: Mother enrichment program (MEP)
Cre-EBD78
SUMO-conjugating enzyme
Activator of the anaphase-promoting complex (APC)
Arrest in M-phase
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Results: Mitochondrial dysfunction in aged yeast
Calcofluor staining of the bud scar
TOM70-GFPMitochondrial marker
Nr. Cell division
Characterization of age-associated mitochondrial phenotypes
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Results: Decreased of mitochondrial function
DiOC6 : ΔΨ-dependent mitochondrial fluorescent dye
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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
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Results: VAM1 and VPH2
VAM1 VPH2
A Catalytic subunit of V-ATPase
V-ATPase assemblyV-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
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Results: Vacuolar and mitochondrial function are linked
• Overexpression of VMA1 and VPH2 increase both media and maximal replicative lifespan.
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How vpH affects mitochondrial function?
vacuolar protein degradation?
Deletion of PEP4
No alteration of mitochondrial structure
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How vpH affects mitochondrial function?
Import of vacuolar metabolite?
Overexpression of vacuolar importers
+ConcA
ATV1 Neutral amino acid transporterPrevent mitochondrial dysfunction
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Results: ATV1 prevents mitochondrial fragmentation
Overexpression of ATV1
• Prevent alteration of mitochondrial structure• No change in vacuolar acidification
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Results: ATV1 as aging modulator
Deletion of ATV1
Development of age-induced mitochondrial dysfunction
Overexpression = Extending RLS
Deletion = Shortening of RLS
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Conclusion
o Yeast is a good model for agingo Link between vacuolar pH and mitochondrial functiono Link between metabolite transport and agingo Vacuole functionalion as aging modulatoro Reestablishment of acidification in the vacuole in the
daughter cells
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Thank you