Budding yeast cells start out with syntelic attachment

1
Budding yeast cells start out with syntelic
attachment and then correct it
2
Mutations in the kinase Ipl1 (Aurora B) result in
mono-oriented sister chromatids
(40 of cells)
(60 of cells)
Tanaka et al. (2002) Cell 108, 317.
3
Budding yeast cells start out with syntelic
attachment and then correct it
Ipl1
4
The Ipl1 homolog Aurora B is required for
correction of syntelic attachments in vertebrate
cells too
treat with monastrol, inhibitor
of centrosome separation
(a) no further treatment (control)
monopolar, syntelic attachments
wash out monastrol
or
(b) add Aurora inhibitor
control
Aurora inhibitor
Lampson et al (2004) Nat. Cell Biol. 6 232
5
Models of kinetochore control by Ipl1/Aurora B
Tanaka et al. (2002) Cell 108, 317.
6
Phosphorylation of Ndc80 by Aurora B reduces
microtubule binding affinity
7
Aurora B lags behind when things get tense
PP1
Aurora B
PP1
Aurora B
Andrews et al (2003) Curr. Opin. Cell Biol. 15
672
PP1
High tension
Low tension
8
Aurora B lags behind when things get tense
Low tension Ndc80 phosphorylated, low
microtubule binding affinity
Aurora B
9
Aurora B lags behind when things get tense
High tension Ndc80 dephosphorylated, high
microtubule binding affinity
Aurora B
10
Basic framework of the cell-cycle control system
Cdc20
11
Securin and cyclin B1 are destroyed in metaphase
Hagting et al (2002) J. Cell Biol. 157 1125
12
Stabilized mitotic cyclins block the events of
late mitosis
Stabilized Cyclin B mutant
Spindle disassembly in telophase
Nuclear envelope assembly
Cytokinesis
Anaphase chromosome movement, spindle stability
Chromosome decondensation
13
Stabilized securin mutant blocks sister separation
Hagting et al (2002) J. Cell Biol. 157 1125
14
Securin is the key APCCdc20 target in sister
separation
15
Securin binds to Esp1
16
The Eureka! experiment
securin
Scc1
SUsupernatant
CPchromatin pellet
Uhlmann et al (1999) Nature 400 37
17
Scc1 cleavage occurs during anaphase
Uhlmann et al (1999) Nature 400 37
18
Non-cleavable Scc1 blocks sister separation
budding
sister separation
Uhlmann et al (1999) Nature 400 37
19
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
20
The story so far
Scc1 cleavage
Sister separation
21
The grand summary of late mitotic control as we
know it
APCCdc20
Cyclin-Cdk
securin
separase
cohesin
spindle disassembly, mitotic exit
sister segregation
sister separation
sister chromatids
metaphase
anaphase
telophase
22
Maintenance of chromosome positioning through
cell division
H2B-CFP
H2B-YFP
merge
pre- bleach
image processing
post- bleach
Gerlich et al (2003) Cell 112 751
23
Maintenance of chromosome positioning through
mitosis
Gerlich et al (2003) Cell 112 751
24
Sister pairs separate in a specific sequence
CENP-A in green - Histone H2B in red
Gerlich et al (2003) Cell 112 751
25
Measuring sister behavior in budding yeast
20 s
110 s
chromosome IV always separates before chromosome V
Holt et al. Nature 454, 353.
26
What mechanism underlies the ordering of sister
separation?
APCCdc20
securin
separase
cohesin
sister segregation
sister separation
sister chromatids
metaphase
anaphase
27
What gets it all started?
28
What determines the timing of APC target
destruction?
Spindle checkpoint
29
The spindle checkpoint system
30
Finding components of the spindle checkpoint
system
and the winners are Mad1, Mad2, Mad3, Bub1,
Bub3, et al