When Old Mothers Go Bad: Replicative aging in budding yeast cells Dr. Michael McMurray Dept....
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When Old Mothers Go Bad: Replicative aging in
budding yeast cells
Dr. Michael McMurrayDept. Molecular & Cell Biology
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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
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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
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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?
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?
The symmetry of cell division and replicative aging
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“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
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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)
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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
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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”…
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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
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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…
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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
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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)
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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
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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?
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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
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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
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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
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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
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How does Yeast Aging relate to Cellular Senescence in Humans?
• Telomere-independent
• Asymmetrically dividing cells
• For what cell type is this a model?
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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
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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