hagting et al (2002) j. cell biol. 157: 1125 securin and cyclin are destroyed in metaphase
TRANSCRIPT
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Hagting et al (2002) J. Cell Biol. 157: 1125
Securin and cyclin are destroyed in metaphase
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Securin binds to Esp1
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The “Eureka!” experiment
SU=supernatant CP=chromatin pellet
Scc1
securin
Uhlmann et al (1999) Nature 400: 37
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Uhl
ma
nn e
t al
(19
99)
Nat
ure
400
: 3
7
Scc1 cleavage occurs during anaphase
Non-cleavableScc1 mutant
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Uhlmann et al (2000) Cell 103: 375
The fabulous Scc1-TEV experiment
Induce TEV expression
Arrest cells in metaphaseby shutting off
CDC20
Measure anaphase onset
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Scc1cleavage
Sisterseparation
The story so far
Separase
Separase
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Stabilized Cyclin mutant
Spindledisassemblyin telophase
Nuclearenvelopeassembly
Chromosomedecondensation
CytokinesisAnaphasechromosomemovement,spindle stability
Anaphase depends on cyclin destructionand Cdk substrate dephosphorylation
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Murata-Hori et al. (2002) Mol. Biol. Cell 13:1099.
GFP-Aurora Bin
wild-typecells
GFP-Aurora Bin
cells withstabilizedCyclin B
Aurora B is removed from kinetochores in early anaphase
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Oliveira et al (2010) Nature Cell Biol. 12:185
Wild-type mitosis TEV-induced anaphase TEV-induced anaphase
TEV-inducedanaphase
+Cdk inhibitor
Wild-typemitosis
The fabulous Scc1-TEV experiment, in flies
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cohesin
separase
securin
Cyclin-CdkAPCCdc20
spindledisassembly,mitotic exit
sisterchromatids
metaphase anaphase telophase
sistersegregation
sisterseparation
Cdk substratedephosphorylation
The grand summary of late mitotic control as we know it
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cohesin
separase
securin
Cyclin-CdkAPCCdc20
spindledisassembly,mitotic exit
sisterchromatids
metaphase anaphase telophase
sistersegregation
sisterseparation
Cdk substratedephosphorylation
More refinements in late mitotic control: budding yeast
Cdc14phosphatase
Holt et al. (2008) Nature 454, 353.
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cohesin
separase
securin
Cyclin-CdkAPCCdc20
spindledisassembly,mitotic exit
sisterchromatids
metaphase anaphase telophase
sistersegregation
sisterseparation
Cdk substratedephosphorylation
More refinements in late mitotic control: human cells
Shindo et al. (2012) Dev. Cell 23, 112.
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What determines the timing of APC target destruction?
“Spindle assembly checkpoint”
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The spindle assembly checkpoint
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Finding components of the spindle checkpoint
…and the winners are: Mad1, Mad2, Mad3, Bub1, Bub3, et al.
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Waters et al (1998) J. Cell Biol. 141: 1181
Mad2 at unattached kinetochores
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Mad1-Mad2complex atunattachedkinetochore
Mad2Mad1
Unattached kinetochores catalyze the formation ofMad2-Cdc20 complexes
Bub3Mad3
Mad2 Cdc20
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Open Mad2(O-Mad2)
Mad2 comes in two structural flavors
Closed Mad2(C-Mad2)
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The Mad1-Mad2 complex at the unattached kinetochore
Closed Mad2(C-Mad2)
Closed Mad2(C-Mad2)
Mad1
Mad1
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Open Mad2(O-Mad2)
Closed Mad2(C-Mad2)
Mad1 or Cdc20
Mad2 uses the safety belt to bind Mad1 or Cdc20
Intermediate Mad2(I-Mad2)
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Mapelli et al. (2007) Cell 131: 730
Mad1
C-Mad2template
Mapelli et al. (2006) EMBO J. 25: 1273
Mapelli and Musacchio (2007)Curr. Opin. Struct. Biol. 17: 716
Mad1
C-Mad2template
C-Mad2copy
Cdc20
Cdc20
The Musacchio Mad2 template model
De Antoni et al. (2005) Curr. Biol. 15, 214
O-Mad2
Mad1
I-Mad2 C-Mad2template
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E2SSNH2
N
The Anaphase-Promoting Complex: E3 extraordinaire
ActivatorSubunitCdc20
Substrate(e.g. securin)
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Cdc20
Mad2
Bub3Mad3
E2
Mad2 collaborates with Mad3-Bub3 to lock down inactive Cdc20
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Schreiber et al. Nature 470, 227 (2011)
David Barford Phil. Trans. Roy. Soc. London B 366, 3605 (2011)
Apc2
Apc11
A
Cdc27
Cdc23
Cdc16
Doc1
Doc1
Apc2
Apc11
Cdc27
A(Cdh1)
Cdc20or
Cdh1
APC under the EM
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Schreiber et al. Nature 470, 227 (2011)
David Barford Phil. Trans. Roy. Soc. London B 366, 3605 (2011)
Doc1
Apc2
Apc11
SA
Cdc27
Cdc23
Cdc16
Doc1
APC under the EM
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Doc1
Cdc20 Cdc20
Mad2-Mad3-Bub3
Herzog et al. Science 323, 1477 (2009)
Cdc20 is repositionedin the spindle checkpoint arrest
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P P
P
P
P
PP
Aurora B
Low tension: Ndc80 phosphorylated, low microtubule binding affinitySpindle checkpoint on
Mad1-Mad2 complex is recruited to apoorly attached (phosphorylated?) kinetochore
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Mis-localizing Aurora B at the centralor outer kinetochore causes anaphase delays
(Inner kinetochore)
(Central kinetochore)
(Outer kinetochore)
Liu et al. (2009)Science 323, 1350
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What determines the timing of APC target destructionin flies and yeast?
Spindle checkpoint
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Maintenance of chromosome positioning through cell division
H2B-CFP H2B-YFP merge
pre-bleach
post-bleach
imageprocessing
S phase G2 to metaphase
Gerlich et al (2003) Cell 112: 751
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Gerlich et al (2003) Cell 112: 751
Maintenance of chromosome positioning through cell division
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Sister pairs separate in a specific sequence
CENP-A in green - Histone H2B in red
Gerlich et al (2003) Cell 112: 751