Towards the architecture of the human inner kinetochore [Elektronische Ressource] / von Sandra Orthaus

Sorthaus

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Informations

• Publié par: friedrich-schiller-universitat_jena
• Publié le: 01 janvier 2007
• Nombre de lectures: 21
• Langue: English
• Poids de l'ouvrage: 4 Mo

Extrait

Towards the architecture of the human
inner kinetochore



Dissertation



zur Erlangung des akademischen Grades

doctor rerum naturalium
(Dr. rer. nat.)


vorgelegt dem Rat der Biologisch-Pharmazeutischen Fakultät
der Friedrich-Schiller-Universität Jena


von
Diplom-Biologin Sandra Orthaus
geboren am 02. Juli 1974 in Jena I
Table of contents

1 1 Introduction
1.1. The centromere kinetochore complex 1
1.1.1. The centromere 1
1.1.2. Inner kinetochore proteins 2
1.1.2.1. CENP-A 3
1.1.2.2. CENP-B 4
1.1.2.3. CENP-C 5
1.1.2.4. CENP-H 5
1.1.2.5. CENP-I 6
1.1.3. Outer kinetochore proteins 6
1.2. The nucleosome 7
1.3. Objective 10
12 2 Materials and Methods
2.1. Materials 12
2.1.1. Chemicals 12
2.1.2. Standarts and Kits 12
2.2. Methods 12
2.2.1. Cell culture and transfection into HEp-2 cells 13
2.2.2. Analysis of the CENP-H genotype of HEp-2 cells 13
2.2.3. RNA interference 14
2.2.4. Cell viability assays 15
2.2.5. Cell cycle analysis and cell synchronisation 15
2.2.6. Antibodies and immuno-fluorescence 16
2.2.7. Confocal microscopy 16
2.2.8. Western Blots 17
2.2.9. Plasmids and cloning 17
2.2.10. Förster resonance energy transfer (FRET) 20
2.2.10.1. Acceptor Photobleaching based FRET measurements 24
2.2.10.2. FLIM (Fluorescence Lifetime Measurements) 25
29 3 Results
3.1. RNAi knock down of the human kinetochore protein CENP-H 29
3.1.1. The siRNA led to depletion of CENP-H in human HEp-2 cells 30
3.1.2. Depletion of CENP-H in human cells resulted in aberrant mitotic 32
phenotypes and decreased numbers of living cells but did not lead
to mitotic arrest II
3.1.3. CENP-H depleted kinetochores showed an unchanged presence of 36
the checkpoint protein hBubR1 and a reduced presence of CENP-C
and CENP-E
3.2. Interaction studies within the human kinetochore in living human cells 40
3.2.1. FRET measurements using the acceptor photobleaching method 43
3.2.1.1. Controls 43
3.2.1.2. Interaction studies with the inner kinetochore protein CENP-A 46
3.2.1.3. Interactions between the inner kinetochore proteins CENP-B, 50
CENP-C and CENP-I
3.2.1.4. Analysis of H1.0 interactions at human centromeres 52
3.2.2. FLIM based FRET measurements 55
3.2.2.1. Controls 55
3.2.2.2. Interaction studies with the inner kinetochore protein CENP-A 58
3.2.2.3. Interactions between the inner kinetochore proteins CENP-B, 67
CENP-C and CENP-I
3.2.2.4. Analysis of H1.0 interactions at human centromeres 71
76 4 Discussion
4.1. Functional analysis of the inner kinetochore protein CENP-H 76
4.2. Interaction studies of inner kinetochore proteins by FRET in vivo 79
4.2.1. Assembly of the inner kinetochore proteins CENP-A and CENP-B in 79
living human cells
4.2.2. The inner kinetochore proteins CENP-B, CENP-C and CENP-I assemble 84
to stabilise a centromere-specific chromatin structure in living human
cells
4.2.3. Linker histone H1.0 is present at human centromeres 88
4.2.4. Model of the interface between centromeric chromatin and the inner 91
kinetochore sub-complex
4.3. Future perspectives 93
95 5 Summary
97 6 Zusammenfassung
99 7 References

III
List of figures and tables


Figure 1.1: The human centromere/kinetochore complex. 2
Figure 1.2: Nucleosome core particle. 8
Figure 2.1: Perrin-Jablonski diagram of FRET process and determination of the 21
FRET efficiency through the fluorescence lifetime of the donor.
Figure 2.2: Comparison of FRET and AB-FRET (acceptor bleaching). 23
Figure 2.3: Potential association states between proteins within the centromere 25
kinetochore complex and the respective fluorescence decays.
Figure 2.4: Principle of TCSPC (time correlated single photon counting). 26
Experimental set-up for the FLIM measurements. 27 Figure 2.5:
Figure 3.1.1: Splice variants of the CENP-H gene and position of the siRNA target 30
sequence within the area of exon 2 and 3.
Figure 3.1.2: CENP-H reduction by RNAi did not influence the cellular content of 31
the kinetochore proteins CENP-C, CENP-E and hBubR1.
Figure 3.1.3: Depletion of CENP-H lead to aberrant mitotic phenotypes. 32
Figure 3.1.4: CENP-H resulted in a decreasing growth rate. 33
CENP-H deficient cells displayed an increased number of aberrant Figure 3.1.5: 34
mitotic phenotypes such as misaligned chromosomes and multipolar
spindles.
Figure 3.1.6: Cell cycle analysis revealed no mitotic arrest in CENP-H depleted 35
cells.
Figure 3.1.7: Kinetochores depleted of CENP-H showed an aberrant distribution or 38
lack of CENP-E but still contained CENP-C and hBubR1.
Figure 3.1.8: CENP-H deficient kinetochores contained a decreased amount of 39
CENP-C and CENP-E and about half of the misaligned
chromosomes totally failed to recruit CENP-E.
Figure 3.2.1: Cerulean and EYFP fusion constructs are expressed as full length 42
proteins in human HEp-2 cells.
Figure 3.2.2: AB-FRET controls showed no false negative or positive FRET. 44
IV
Acceptor bleaching in vivo lead to FRET between CENP-B- 48 Figure 3.2.3:
Cerulean/EYFP-CENP-A, Cerulean-CENP-A/EYFP-CENP-A and
Cerulean-CENP-A/EYFP-H4.A
Figure 3.2.4: Acceptor bleaching FRET measurements of the inner kinetochore 51
proteins CENP-B, CENP-C and CENP-I in vivo.
Figure 3.2.5: Acceptor bleaching FRET in vivo revealed the presence of linker 54
histone H1.0 at human centromeres.
Figure 3.2.6: FLIM controls. Fluorescence lifetime imaging of cells (co-) 57
expressing Cerulean and Cerulean-YFP fusion constructs.
Figure 3.2.7: Confocal micrographs and fluorescence lifetime measurements of 60
single HEp-2 cells co-expressing Cerulean and EYFP fusion proteins.
Figure 3.2.8: Lifetime histogram of all kinetochores evaluated in this study. 62
FLIM measurements in vivo to assess the interactions between the 69 Figure 3.2.9:
inner kinetochore proteins CENP-B, CENP-C and CENP-I.
Figure 3.2.10: FLIM in vivo confirmed the association between histone H1.0 and 72
the inner kinetochore proteins CENP-A, -B and C.
Figure 4.1: Schematic model representation of the FRET experiments including 81
CENP-A, CENP-B and core histones.
Figure 4.2: Linear model of the centromere array formed by interactions of the 87
inner kinetochore proteins CENP-B and CENP-C.
Figure 4.3: Binding of linker histone H1.0 to centromeric chromatin. 89
Figure 4.4: Linear model of the centromere organisation and inner kinetochore 92
architecture.
Table 1.: Overview of the AB-FRET and FLIM results. 74
V
List of abbreviations


% percentage
A adenine, alanine
aa amino acid
AB-FRET acceptor bleaching fluorescence resonance energy transfer
ACA anti centromere antibody
APC anaphase promoting complex
ATCC american tissue culture collection
bp base pair
BP band pass filter
BSA bovine serum albumine
°C degree
C cytosine, carboxy terminus
CAD CENP-A-nucleosome distal centromere components
cDNA complementary DNA
CENP centromere protein
C. elegans Caenorhabditis elegans
CREST Calcinosis (cutis), Raynaud (syndrom), Esophageal (dysmotility),
Sklerodactyly, Telangiectasia
CY3 Indocarbocyanin
D aspartic
DAPI 4',6-diamidino-2-phenylindole
DIC differential interference contrast
DMEM Dulbeccos modified Eagles medium
DNA deoxyribonucleic acid
dT deoxy-thymidine
E glutamic
E FRET efficiency f
ECFP enhanced cyan fluorescent protein
ECL enhanced chemiluminescence
E. coli Escherichia coli
EDTA ethylene diamine tetra acetic acid
E efficiency of FRET f VI
e.g. for example (exempli gratia)
EGFP enhanced green fluorescent protein
EYFP enhanced yellow fluorescent protein
et al. et alii
EtBr ethidium bromide
EtOH ethanol
F phenylalanine
FACS fluorescence associated cell sorting
f.e. for example
FCS fetal calf serum
Fig figure
FITC fluorescein isothiocyanate
FLIM fluorescence lifetime imaging
FRET fluorescence resonance energy transfer
FWHM full width half maximum
G guanine, glycine
g gram
Hec1 highly expressed in cancer
HEp-2 human epithelial cell line 2
HCl hydrochloric acid
HP1 heterochromatin protein 1
HRP horse raddish peroxidase
I isoleucine
IgG immune globulin
IIF in direct immuno fluorescence
K lysine
k rate constant
k rate constant for a radiative (fluorescent) process f
k rate constant for a nonradiative process nr
k rate constant for energy transfer t
kb kilo base pairs
kDa kilo Dalton
L leucine
l litre VII
LBO lithium triborate
LP long pass filter
LSM laser scanning microscope
µ micro
m milli
M molar
MAC mammalian artificial chromosome
MCAK mitotic centromere-associated kinesin
MCP-PMT multichannel-plate photomultiplier tube
min minute
hMis12 (human) minichromosome instability
MT microtubules
N amino terminus, asparagin