Sister-chromatid cohesion makes it easy to biorient
With
sister cohesion
Without
sister cohesion
Sister-chromatid cohesion by
DNA catenation
Cohesin is for cohesion
Smc1-Smc3 dimer
Smc1-Smc3 dimer + Scc1 and Scc3
Cutting Scc1 here triggers sister separation in anaphase
How does cohesin hold sisters together?
cutting
Scc1
here
triggers
sister
separation
Testing for topological interactions between cohesin and
a circular minichromosome
sucrose
gradient
native agarose gel
Ivanov and Nasmyth (2007) Mol. Cell 27, 300
Minichromosome dimers are held together by cohesin
Ivanov and Nasmyth (2007) Mol. Cell 27, 300
The latest test of the two major models: Haering et al., 2008
If it’s physical contact
If it’s a topological link
crosslink
protein
ends
crosslink
protein
ends
denature
monomer circles
denature
monomer circles
monomer circles
dimers!
Putting on cohesin
Loading
Scc2/4
+
Cohesin ATPase
activity
Establishment
DNA replication
Eco1 Wpl1
G1
S phase
G2 and M
Affinity of cohesin for chromatin
iFRAP of
GFP-tagged
cohesin
subunit
SA1
Scc3
Scc1
Scc3
Gerlich et al (2006) Curr. Biol. 16, 1571
Affinity of cohesin for chromatin
Gerlich et al (2006)
Curr. Biol. 16, 1571
How does cohesin get loaded?
Gruber et al. (2006) Cell 127, 523.
Does cohesin loading require opening the SMC-Scc1 connection?
Normal
function!
Normal
function!
Normal
function!
Gruber et al. (2006) Cell 127, 523.
Replacing the SMC hinge
blocks cohesin binding to chromosomes
Gruber et al. (2006) Cell 127, 523.
The balance of cohesin loading, release and establishment
Loading
Establishment
Scc2-4
+ ATP
Eco1
Ac
Wpl1
DNA
replication
Chan et al. (2012)
Cell 150, 961.
Wpl1
Wpl1
Release
Wpl1
G1
S
Release
Go through the hoop, or flip it over?
Sister chromatid condensation and resolution
Sumner, A.T.
Chromosoma
1991, 100:410
We know next to nothing about chromosome condensation
400-700 nm
30 nm
Belmont Curr. Opin. Cell Biol. 2006, 18:632
Condensin is required for normal mitotic chromosome
assembly in Xenopus extracts
Control antibody
Anti-condensin antibody
Hirano and Mitchison Cell 1994, 79:449-458
The Condensin complex
Condensin at the chromosome axis in metaphase
DAPI
Smc2
DAPI
Topo II
Topo II
Smc2
Fluorescence
microscopy
Electron
microscopy:
immunogold
of Smc2
Kireeva et al.
J. Cell Biol.
2004, 166:775
How does condensin organize chromosomes?
Glue
Ring
Floral
arrangement
Cuylen and Haering
Trends Cell Biol. 2011, 21:552
Evidence for a condensin ring!
Bgl II
TEV
protease
Cuylen et al. Nat. Struct. Mol. Biol. 2011, 18:894
Artificial condensin ring cutting makes the sister chromatids floppier
Cuylen et al. Nat. Struct. Mol. Biol. 2011, 18:894
Condensin is not required for condensation in human cells (?!)
Control
RNAi
CAP-D2
(condensin I)
RNAi
CAP-D3
(condensin II)
RNAi
SMC2
(both condensins)
RNAi
CAP-D2
& CAP-D3
RNAi
isotonic
treatment
hypotonic
treatment
Nocodazole,
hypotonic
treatment
Hirota et al. J. Cell Sci. 2004, 117:6435
Condensin is required for normal anaphase
Control
RNAi
CAP-D2
(condensin I)
RNAi
CAP-D3
(condensin II)
RNAi
SMC2
(both
condensins)
RNAi
Hirota et al. J. Cell Sci. 2004, 117:6435
Condensin might also help organize sisters for decatenation
by topoisomerase II
Topo II
Cuylen and Haering
Trends Cell Biol. 2011, 21:552
The ‘Prophase pathway’: In animal cells,
cohesin is removed from chromosome arms in early mitosis
control
Polo kinase
removed
Aurora B kinase
removed
Both kinases
removed
DNA
cohesin
Losada et al. Genes Dev. 2002, 16:3004
Cohesin removal from arms is required for sister resolution
control
Both kinases
removed
Losada et al.
Genes Dev. 2002
16:3004
Phosphorylation of a cohesin subunit is required for removal
of arm cohesin in prophase
SA2 in humans
[wild type]
[Nonphosphorylated mutant]
Hauf et al. PLoS Biol. 2005, 3:e69
We still know next to nothing about mitotic chromosome structure
1.
Compaction of DNA from same sister
2.
Resolution of DNA strands from different sisters
3.
Coordination of condensin and cohesin
4.
How do Cdks regulate all this?
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