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