The many functions of the kinetochore
Microtubule
-
- attach sister chromatid to microtubule plus end
- remain attached when microtubule is growing
and shrinking
- allow regulation of attachment to ensure
biorientation
growing
shrinking
- send signals that block anaphase
when attachment is incorrect
+
- generate force to move chromosomes
Kinetochore
Centromere DNA
inner
outer
Kinetochores have many parts
budding yeast
animals
Spc24
Ndc80
Spc25
Nuf2
microtubules
microtubules + Ndc80 complex
Tanaka and Desai Curr. Opin. Cell Biol. 2008, 20:53
The Ndc80 complex links microtubules to inner kinetochore
Ciferri et al. Cell 2008, 133:427
The Ndc80 complex links microtubules to inner kinetochore
The yeast Dam1/DASH complex forms rings around microtubules
Westermann et al. Mol. Cell 2005, 17:277
J.J. Miranda et al. Nat. Struct. Mol. Biol. 2005, 12:138
The yeast Dam1/DASH complex forms rings around microtubules
Ramey et al. (2011) Mol. Biol. Cell 22, 4335.
Joglekar et al. Nature 2010, 463:446
Joglekar et al. Curr. Biol. 2009, 19:694
The yeast kinetochore at super-resolution
The kinetochore of higher eukaryotes
Metaphase
Anaphase
3-D close-up of the animal kinetochore: a meshwork without rings!
Dong et al. Nat. Cell Biol. 2007, 9:516
Santaguida and Musacchio. EMBO J. 2009, 28:2511
The animal kinetochore
Dong et al. Nat. Cell Biol. 2007, 9:516
Cheeseman et al. Cell (2006) 127:983.
EM tomography
of the animal
kinetochore
McIntosh et al.
Cell (2008) 135:322.
metaphase
anaphase
McIntosh et al. Cell (2008) 135:322.
Fibrils link kinetochore to depolymerizing MT ends
Using plus-end depolymerization
to do the work of chromosome movement
Biased diffusion
Forced walk
Joglekar et al. Curr. Opin. Cell Biol. 2009, 22:1
Only the correct bivalent attachment is stable
Bivalent attachment creates tension
that separates the kinetochores
Budding yeast cells start out with syntelic attachment
and then correct it
Mutations in the kinase Ipl1 (Aurora B)
result in mono-oriented sister chromatids
(40% of cells)
Tanaka et al. (2002) Cell 108, 317
(60% of cells)
Models of kinetochore control by Ipl1/Aurora B
Tanaka et al. (2002) Cell 108, 317
Phosphorylation of Ndc80 by Aurora B
reduces microtubule binding affinity
P
PP
P
P
P
Aurora B lags behind when things get tense
Aurora B
PP1
Aurora B
PP1
Low tension
High tension
Andrews et al (2003) Curr. Opin. Cell Biol. 15: 672
PP1
Aurora B lags behind when things get tense
Low tension: Ndc80 phosphorylated, low microtubule binding affinity
P
P
Phosphatase PP1
P
P
Aurora B
P
P
P
Aurora B lags behind when things get tense
High tension: Ndc80 dephosphorylated, high microtubule binding affinity
Aurora B
FRET-based Aurora B activity sensor
Aurora B
TFP
High
YFP:TFP
ratio
TFP
Low
YFP:TFP
ratio
Violin et al. (2003) J. Cell Biol. 161, 899
Fuller et al. (2008) Nature 453, 1132
Liu et al. (2009) Science 323, 1350
Dephosphorylation of Aurora B sensor
in a tense kinetochore
CENP-B
Mis12
Liu et al. (2009) Science 323, 1350
Mis-localizing Aurora B at the central or outer kinetochore
causes mitotic defects
(Inner kinetochore)
(Central kinetochore)
(Outer kinetochore)
Liu et al. (2009)
Science 323, 1350
Securin and cyclin are destroyed in metaphase
Hagting et al (2002) J. Cell Biol. 157: 1125
Stabilized securin mutant blocks sister separation
(APC activator)
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