1. When Old Mothers Go Bad: Replicative aging in budding yeast cells
Dr. Michael McMurray
Dept. Molecular & Cell Biology

2. Outline Intro to yeast aging A molecular cause of yeast aging
SIR2: A conserved regulator of longevity?
Aging and genetic instability, in yeast and humans

3. Cellular senescence: finite replicative capacity of mitotically dividing cells
Originally observed in human diploid fibroblasts (Hayflick limit, 1965)
Represents a limit on the number of population doublings
Caused by telomere shortening in cells that do not express telomerase

4. What about simple eukaryotic cells that do express telomerase?
Cells of baker’s yeast, Saccharomyces cerevisiae, express telomerase
Microbial populations are “immortal”, can be passaged forever
Does this mean these cells are also immortal?

5. The symmetry of cell division and replicative aging?

6. The Cell Spiral Model of Yeast Aging
Generation
(cell cycle)
“virgin” cell
• Sterility
• increased size
• wrinkles
• bud scars
• increased generation time
AGING
Lifespan = n (20-40)
Adapted from Jazwinski, et al Exp Geront 24: (1989)
nth
daughtern
daughter1
1st
dead cell
(lysis)

7. How does the population remain immortal?
In every daughter cell, the lifespan “clock” is reset to zero
Each division produces a cell that can divide many more times
Senescent cells are very rare in a large, exponentially growing population (1/2a+1)

8. What is the role of telomere length in yeast cellular senescence?
Telomerase is expressed throughout the lifespan
Telomere length is maintained throughout the lifespan
Mutating telomerase does cause cellular senescence: telomere shortening, limited population doublings, genomic instability, ALT

9. What causes yeast aging?
A clue: exceptions to the rule of the resetting clock
Occasionally, daughters of old mothers are born prematurely aged!
Their lifespan equals the mother’s remaining lifespan
The asymmetry has broken down -- accompanied by loss of size asymmetry (“symmetric buds”)
The daughters of symmetric buds have normal lifespan
Suggests these symmetric buds have inherited a “senescence factor”…

10. The Yeast Senescence Factor Model (1989)
Preferentially segregated to mother cell each division
Accumulates to high concentrations in old mothers
Eventually inhibits cell division, causes other aging phenotypes
Is occasionally inherited by symmetric buds

11. What is the yeast senescence factor?
Some clues (late 1990s):
Aging is accompanied by fragmentation of the nucleolus
The nucleolus assembles at the site of rRNA transcription, the rDNA
Sir2 localizes to the nucleolus, and sir2 mutants have a short lifespan
sir2 mutants have high levels of extrachromosomal rDNA circles (ERCs)
ERCs have the characteristics of the senescence factor…

12. Extrachromosomal rDNA Circles as a cause of yeast aging
Excised from the chromosomal array by recombination
Recombination is suppressed by Sir2
Replicate nearly every cell cycle
Have a strong mother segregation bias at mitosis
High levels can inhibit cell division
Inherited by the daughters of old mothers

13. But, no ERCs in humans! (or mice, or worms, or flies…) Why continue to study yeast aging?
Overexpressing SIR2 homologs in flies and worms extends lifespan
Perhaps the regulation of lifespan is conserved (and SIR2-dependent) while the molecular effectors of aging vary between organisms
Example: calorie restriction (CR)

14. Calorie Restriction (CR) Extends Lifespan
Decreasing caloric intake (without starvation) lengthens lifespan
Works in yeast, flies, rats, mice, worms, …
Many reports claimed that the CR pathway is SIR2-dependent, supporting theory of SIR2 as master aging regulator
Heated debate over the mechanism by which SIR2 influences CR pathway
Recent work has shown that in some yeast strains CR is actually SIR2-independent

15. Genetic instability and Aging
Frequencies of mutations and chromosomal rearrangements increase with age in various organisms
Incidence of cancer increases dramatically with age:
Is this due to accumulation of genetic events at a constant rate over the lifetime, or does aging itself alter the rate of new genetic events?

16. Yeast pedigree analysis
Separate daughter from mother
Instead of discarding, isolate daughters
Let daughters form colonies
Assay for Loss of Heterozygosity (LOH)
Change in rate during lifespan?
LOH
wildtype
mutant

17. An Age-induced Hyper-recombinational State
humans
yeast
After about 25 divisions, aging mother cells begin to produce daughters that are genetically unstable
High rates of LOH at multiple chromosomes
LOH is caused by recombination, not chromosome loss or deletion
Behaves as a “switch” to a new, unstable state
Hyper-recombinational state is eventually “diluted” in progeny of old cells

18. This is reminiscent of the Yeast Senescence Factor!
Something accumulates with each cell division in mother
Reaches a threshold, causes genetic instability
Inherited by daughters of old mothers
Eventually “reset” in distant progeny

19. Are ERCs the cause?
Mutations that increase ERCs (sir2) do not accelerate onset of switch
Mutations that decrease ERCs do not delay onset of switch
In fact, onset of switch is unlinked to lifespan!
Suggests an important distinction between longevity and functional senescence

20. How does Yeast Aging relate to Cellular Senescence in Humans?
Telomere-independent
Asymmetrically dividing cells
For what cell type is this a model?

21. Stem cells in human aging and cancer
Evidence that stem cells are important in aging and cancer
Immunological senescence
“Cancer stem cells”
Stem cells often express telomerase
Stem cells divide asymmetrically

22. Conclusions
Yeast aging involves longevity regulation as well as senescence phenotypes unlinked from longevity
Genetic instability increases with age in yeast, by an epigenetic hyper-recombinational switch
May be a good model for stem cell aging