The human kinetochore
Stephan Diekmann
S. Diekmann
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EUKARYOTIC CELL CYCLE
Prometaphase
Interphase
Prophase
DNA
synthesis
G2
mitosis
Metaphase
Telophase
cytokinesis
Anaphase
G1
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(modified Cleveland et al., 2003)
HUMAN KINETOCHORE
Cleveland et al. Cell 2003
Centromere: DNA, chromatin
S. Diekmann
Kinetochore: multi-protein complex
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KINETOCHORE FUNCTION
low copy number plasmids
Prokaryotes:
mechanic link
- 1 genome
- no control required
Actin-like
structure
centromere
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Mechanistic working principle
Strong forces applied to a long thin molecule (DNA)
 DNA must be packaged.
Mechanistic part has 3 elements:
1.
Site on the DNA ( „centromere“)
2.
Protein binds DNA site ( „kinetochore“),
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2 1
DNA packaging
3.
Force receptor (linear; filament, tubulus, ...)
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The principle is realised by
bacterial low copy number
plasmids (E. coli):
parC (DNA)
parR
protein
- specific DNA binding site
- 1 kinetochore complex
- force generator and acceptor:
1 filament parM (actin, pushing)
parM
Moeller-Jensen et al., 2003
 minimal system
(only 2 proteins!)
Prediction: DNA curvature
DNA packaging
complex formation
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Large molecules and complexes are moved
(pushed) through viscous cellular medium:
Hoischen et al., 2008
 high power transmission,
DNA-protein complex must be tight
 many contacts!
 DNA (centromere) packaged into a
complex (kinetochore)!
Pushing movement
nucleosome-like folding
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KINETOCHORE FUNCTION
low copy number plasmids
Prokaryotes:
mechanic link
- 1 genome
- no control required
Actin-like
structure
centromere
Eukaryotes:
- genome distributed
on several
chromosomes
- tight control required
Checkpoint
mechanic link
Mikrotubuli
inner kinetochore
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The most simple eukaryotic centromere/kinetochore:
Yeast
S. cerevisiae
- specific DNA binding site (CDE III),
curved DNA
- modified nucleosome!
- 1 kinetochore complex (many proteins)
- force acceptor: 1 microtubule
CDE III
Pollard, 2002
(1 microtubule pulls about 1 Mbp)
Bechert et al., NAR 27 (1999) 1444
Hemmerich et al., PNAS 97 (2000) 12583
Wieland et al., NAR 29 (2001) 1054
- many packaging proteins (including histones)
- connection to microtubule (adaptor) contains
many checkpoint proteins (controlling 16 chromosomes)
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KINETOCHORE FUNCTION
low copy number plasmids
Prokaryotes:
mechanic link
- 1 genome
- no control required
Actin-like
structure
centromere
Eukaryotes:
- genome distributed
on several
chromosomes
- tight control required
Checkpoint
human
mechanic link
Mikrotubuli
inner kinetochore
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KiNETOCHORE DURING THE CELL CYCLE
Expectation:
Kinetochore proteins assemble in G2 and dissociate in early G1
DNA
synthesis
G2
mitosis
cytokinesis
G1
Observed:
Kinetochore proteins present during the whole cell cycle
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EXPERIMENTAL APPROACH
Dynamics
Structure
RICS
Interaction
FRAP
EM/AFM
Y2H, M2H
Dynamics
in vivo, in situ
cell cycle dependent
proximity
in vitro
cell cycle dependent
FRET
Western blots
Interaction
Pull-down
Mass Spec
FCS
protein
abundance
protein modification
Dynamics
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Nsl1 Dsn1
Mis Nnf1
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CCAN
Q
I
S X U O
K
M R L
T
W
H3 B A
P
N
centromere
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H
C
Chromosome
hMis12Komplex
inner
outer
kinetochore
Y2H (INNER KINETOCHORE)
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M2H kinetochore protein interactions
in the human
cell nucleus:
CENP
collaboration with
Wen Deng
Heinrich Leonhardt
LMU Munich
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M2H kinetochore protein interactions
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Wen Deng
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ANALYSIS OF THE INNER KINETOCHORE (INTERPHASE)
Study of many proteins of the inner kinetochore:
CENP-A, -B, -C
CENP-T, -W, -S, -X
CENP-Q, -U, -P, -O, -R
hMis12 complex: Nnf1, Mis12, Dsn1, Nsl1
 here 1 example: CENP-N
Q
I
S X U O K H
M R L
T
C
W
H3 B A
P
N
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CENP-N protein abundance
A
B
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CENP-N dynamics
FRAP: G1 and early S phase
FRAP: EGFP-CENP-NDC
mid S-phase
RICS: EGFP-CENP-N
cytoplasm
nucleoplasm
kinetochore
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Cell cycle G1 to M
CENP-N DYNAMICS
Mitosis
G1-Phase
S-Phase
G2-Phase
1,0
amount
0,8
loading
0,6
reduction
0,4
loading
0,2
slow
binding
fast exchange
0
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Hellwig et al.,19
2011
CENP-N STABILISATION DURING S-PHASE
interaction shown in vitro
Carroll et al., 2009
CENP-L
C
CENP-L
CENP-L
CENP-N
C
CENP-N
CENP-NDC
CENP-A
CENP-A
G1 and early S-phase
middle and late S-phase
CENP-A
middle S-phase
CENP-N proximity to CENP-A: FRET
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Hellwig et al., 2011
NUCLEOSOME VARIANTS
H3 octameric nucleosome,
classical chromatin nucleosome,
left-handed DNA twist
CENP-A tetrameric nucleosome,
centromere specific nucleosome,
right-handed DNA twist
hint for a centromere specific
chromatin structure
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Dalal et al., 2007
Furuyama, Henikoff, 2009
Dimitriadis et al., 2010
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Structural kinetochore model (Interphase)
Nsl1
Dsn1
- Y2H
Mis12
- M2H
Nnf1
Q
O
I
- FRET
H
L
P
U
- FRAP
S
M
X
C
N
T
W
H3 nucleosome
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K
R
B
CenpA nucleosome
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SUMMARY
Our results yield a molecular (not complete yet)
understanding of the human inner kinetochore
during interphase:
the inner kinetochore
• assembles in S-phase (due to increased
concentrations of several but not all
proteins) in a self-assembly process
• forms a bridge between CENP-A and H3
containing nucleosomes
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ACKNOWLEDGEMENTS
FLI
Galway Irland
Warwick UK
Christian Hoischen
Marlen Stäglich
Tobias Ulbricht
Daniela Hellwig
Sylke Pfeiffer
Steffi Weidtkamp-Peters
Anja Eskat
Volker Döring
Antje Hofmeister
Elke Mies
Christian Weber
Sabine Ohndorf
Sandra Orthaus
Peter Hemmerich
Ivana Sumanovac
Carsten Dornblut
Sven Rudolphi
Sindy Giebe
Indri Erliandri
Britta Reichenbächer
Nadine Quinn
Lisa Prendergast
Chelly van Vuuren
Kevin Sullivan
Andrew McAinsh
Stanford USA
Aaron Straight
Christoph Carroll
NIH USA
Minh Bui
Yamini Dalal
LMU Munich
Wen Deng
Heinrich Leonhardt
FSU Jena
Peter Dittrich
Bashar Ibrahim
Rainer Heintzmann
Martin Westermann
ETH Zürich
Patrick Meraldi
Stephan Emmerth
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AND…
Thank you for your attention!
My dog is named „Manuscript“
since whereever I send him to
he immediately comes back.
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