Securin and cyclin are destroyed in metaphase
Hagting et al (2002) J. Cell Biol. 157: 1125
Securin binds to Esp1
The “Eureka!” experiment
securin
Scc1
SU=supernatant
Uhlmann et al (1999) Nature 400: 37
CP=chromatin pellet
Non-cleavable
Scc1 mutant
Uhlmann et al (1999) Nature 400: 37
Scc1 cleavage occurs during anaphase
The fabulous Scc1-TEV experiment
Arrest cells in metaphase
by shutting off
CDC20
Induce TEV expression
Measure anaphase onset
Uhlmann et al (2000) Cell 103: 375
The story so far
Scc1
cleavage
Separase
Separase
Sister
separation
Anaphase depends on cyclin destruction
and Cdk substrate dephosphorylation
Stabilized Cyclin mutant
Anaphase
chromosome
movement,
spindle
stability
Spindle
disassembly
in telophase
Nuclear
envelope
assembly
Cytokinesis
Chromosome
decondensation
Aurora B is removed from kinetochores in early anaphase
GFP-Aurora B
in
wild-type
cells
GFP-Aurora B
in
cells with
stabilized
Cyclin B
Murata-Hori et al. (2002) Mol. Biol. Cell 13:1099.
The fabulous Scc1-TEV experiment, in flies
Wild-type mitosis
TEV-induced anaphase
TEV-induced anaphase
TEV-induced
anaphase
+
Cdk inhibitor
Wild-type
mitosis
Oliveira et al (2010) Nature Cell Biol. 12:185
The grand summary of late mitotic control as we know it
APCCdc20
Cyclin-Cdk
Cdk substrate
dephosphorylation
securin
separase
cohesin
sister
separation
sister
segregation
spindle
disassembly,
mitotic exit
sister
chromatids
metaphase
anaphase
telophase
More refinements in late mitotic control: budding yeast
APCCdc20
securin
Cyclin-Cdk
Cdk substrate
dephosphorylation
Cdc14
phosphatase
separase
cohesin
sister
separation
sister
segregation
spindle
disassembly,
mitotic exit
sister
chromatids
metaphase
Holt et al. (2008) Nature 454, 353.
anaphase
telophase
More refinements in late mitotic control: human cells
APCCdc20
Cyclin-Cdk
Cdk substrate
dephosphorylation
securin
separase
cohesin
sister
separation
sister
segregation
spindle
disassembly,
mitotic exit
sister
chromatids
metaphase
Shindo et al. (2012) Dev. Cell 23, 112.
anaphase
telophase
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.
Mad2 at unattached kinetochores
Waters et al (1998) J. Cell Biol. 141: 1181
Unattached kinetochores catalyze the formation of
Mad2-Cdc20 complexes
Mad1 Mad2
Mad2 Cdc20
Bub3
Mad1-Mad2
complex at
unattached
kinetochore
Mad3
Mad2 comes in two structural flavors
Open Mad2
(O-Mad2)
Closed Mad2
(C-Mad2)
The Mad1-Mad2 complex at the unattached kinetochore
Mad1
Closed Mad2
(C-Mad2)
Mad1
Closed Mad2
(C-Mad2)
Mad2 uses the safety belt to bind Mad1 or Cdc20
Mad1 or Cdc20
Open Mad2
(O-Mad2)
Closed Mad2
(C-Mad2)
Intermediate Mad2
(I-Mad2)
The Musacchio Mad2 template model
Mad1
Mad1
O-Mad2
C-Mad2
template
I-Mad2
C-Mad2
template
Cdc20
Mad1
Cdc20
De Antoni et al. (2005) Curr. Biol. 15, 214
Mapelli et al. (2006) EMBO J. 25: 1273
Mapelli et al. (2007) Cell 131: 730
Mapelli and Musacchio (2007)
Curr. Opin. Struct. Biol. 17: 716
C-Mad2
copy
C-Mad2
template
The Anaphase-Promoting Complex: E3 extraordinaire
Substrate
(e.g. securin)
S
S
NH2
N
Activator
Subunit
Cdc20
E2
Mad2 collaborates with Mad3-Bub3 to lock down inactive Cdc20
Cdc20
Bub3
Mad3
Mad2
E2
APC under the EM
Cdc20
or
Cdh1
Cdc27
A
(Cdh1)
Cdc27
A
Doc1
Cdc16
Cdc23
Doc1
Apc11
Apc11
Apc2
Apc2
Schreiber et al. Nature 470, 227 (2011)
David Barford Phil. Trans. Roy. Soc. London B 366, 3605 (2011)
APC under the EM
Cdc27
A
Cdc16
Cdc23
S
Doc1
Apc11
Apc2
Schreiber et al. Nature 470, 227 (2011)
David Barford Phil. Trans. Roy. Soc. London B 366, 3605 (2011)
Doc1
Cdc20 is repositioned
in the spindle checkpoint arrest
Cdc20
Cdc20
Doc1
Herzog et al. Science 323, 1477 (2009)
Mad2Mad3Bub3
Mad1-Mad2 complex is recruited to a
poorly attached (phosphorylated?) kinetochore
Low tension: Ndc80 phosphorylated, low microtubule binding affinity
Spindle checkpoint on
P
P
P
P
Aurora B
P
P
P
Mis-localizing Aurora B at the central
or 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 destruction
in flies and yeast?
Spindle checkpoint
Maintenance of chromosome positioning through cell division
H2B-CFP
H2B-YFP
merge
prebleach
image
processing
postbleach
S phase
G2 to metaphase
Gerlich et al (2003) Cell 112: 751
Maintenance of chromosome positioning through cell division
Gerlich et al (2003) Cell 112: 751
Sister pairs separate in a specific sequence
CENP-A in green - Histone H2B in red
Gerlich et al (2003) Cell 112: 751