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When Old Mothers Go Bad: Replicative aging in
budding yeast cells
Dr. Michael McMurrayDept. Molecular & Cell Biology
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
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
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?
?
The symmetry of cell division and replicative aging
“virgin” cell
daughter1
1st
Generation(cell cycle)
dead cell(lysis)
nth
daughtern
• Sterility• increased size• wrinkles• bud scars• increased generation time
AGING
Lifespan = n (20-40)
Adapted from Jazwinski, et al Exp Geront 24:423-48 (1989)
The Cell Spiral Model of Yeast Aging
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)
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
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”…
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
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…
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
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)
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
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?
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
wildtypemutant
An Age-induced Hyper-recombinational State
• 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
humans yeast
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
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
How does Yeast Aging relate to Cellular Senescence in Humans?
• Telomere-independent
• Asymmetrically dividing cells
• For what cell type is this a model?
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
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