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Hagting et al (2002) J. Cell Biol. 157: 1125
Securin and cyclin are destroyed in metaphase
Securin binds to Esp1
The “Eureka!” experiment
SU=supernatant CP=chromatin pellet
Scc1
securin
Uhlmann et al (1999) Nature 400: 37
Uhl
ma
nn e
t al
(19
99)
Nat
ure
400
: 3
7
Scc1 cleavage occurs during anaphase
Non-cleavableScc1 mutant
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
Scc1cleavage
Sisterseparation
The story so far
Separase
Separase
Stabilized Cyclin mutant
Spindledisassemblyin telophase
Nuclearenvelopeassembly
Chromosomedecondensation
CytokinesisAnaphasechromosomemovement,spindle stability
Anaphase depends on cyclin destructionand Cdk substrate dephosphorylation
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
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
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
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.
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.
What determines the timing of APC target destruction?
“Spindle assembly checkpoint”
The spindle assembly checkpoint
Finding components of the spindle checkpoint
…and the winners are: Mad1, Mad2, Mad3, Bub1, Bub3, et al.
Waters et al (1998) J. Cell Biol. 141: 1181
Mad2 at unattached kinetochores
Mad1-Mad2complex atunattachedkinetochore
Mad2Mad1
Unattached kinetochores catalyze the formation ofMad2-Cdc20 complexes
Bub3Mad3
Mad2 Cdc20
Open Mad2(O-Mad2)
Mad2 comes in two structural flavors
Closed Mad2(C-Mad2)
The Mad1-Mad2 complex at the unattached kinetochore
Closed Mad2(C-Mad2)
Closed Mad2(C-Mad2)
Mad1
Mad1
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)
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
E2SSNH2
N
The Anaphase-Promoting Complex: E3 extraordinaire
ActivatorSubunitCdc20
Substrate(e.g. securin)
Cdc20
Mad2
Bub3Mad3
E2
Mad2 collaborates with Mad3-Bub3 to lock down inactive Cdc20
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
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
Doc1
Cdc20 Cdc20
Mad2-Mad3-Bub3
Herzog et al. Science 323, 1477 (2009)
Cdc20 is repositionedin the spindle checkpoint arrest
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
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
What determines the timing of APC target destructionin flies and yeast?
Spindle checkpoint
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
Gerlich et al (2003) Cell 112: 751
Maintenance of chromosome positioning through cell division
Sister pairs separate in a specific sequence
CENP-A in green - Histone H2B in red
Gerlich et al (2003) Cell 112: 751