Mps1-dependent phosphorylation of the kinetochore protein Ndc80 is an intrinsic step in spindle checkpoint activation [Elektronische Ressource] / presented by Stefan Kemmler

Sujets

• Biologie

Informations

• Publié par: ruprecht-karls-universitat_heidelberg
• Publié le: 01 janvier 2008
• Nombre de lectures: 32
• Langue: English
• Poids de l'ouvrage: 20 Mo

Extrait

Mps1-dependent phosphorylation of
the kinetochore protein Ndc80 is an intrinsic step in
spindle checkpoint activation
Dissertation
submitted to the
Combined Faculties for the Natural Sciences and for Mathematics
of the Ruperto-Carola University of Heidelberg, Germany
for the degree of
Doctor of Natural Sciences
presented by
Stefan KemmlerDissertation
submitted to the
Combined Faculties for the Natural Sciences and for Mathematics
of the Ruperto-Carola University of Heidelberg, Germany
for the degree of
Doctor of Natural Sciences
presented by
Diplom-Biologist Stefan Kemmler
born in: Karlsruhe
Oral-examination: 17.12.2007Mps1-dependent phosphorylation of
the kinetochore protein Ndc80 is an intrinsic step in
spindle checkpoint activation
Referees: Prof. Dr. Michael Brunner
PD Dr. Johannes LechnerDiese Arbeit ist meinen Eltern gewidmet.TABLE OF CONTENTS
Table of contens I-IV
Abbreviations V-VI
Summary 00 1
Zusammenfassung 00 3
1 Introduction 00 5
1.1 S. cerevisiae as a model organism 00 5
1.2 The cell cycle 00 5
1.3 Mitotic checkpoints 00 8
1.3.1 The spindle assembly checkpoint (Mad2-checkpoint) 009
1.3.1.1 Microtubule-Attachment 009
1.3.1.2 Kinetochore-Tension 012
1.3.2 The spindle positioning checkpoint (Bub2-checkpoint) 014
1.4 The kinetochore 016
1.4.1 Centromere DNA 016
1.4.2 Kinetochore proteins 017
1.5 Kinetochore structure 025
1.6 Phosphoepitopes at the kinetochore 026
2 Goal of the present work 027
3 Materials 028
3.1 Plasmids 028
3.2 E.coli strains 031
3.3 S. cerevisiae strains 031
3.4 Culturing conditions & Media 037
3.5 Oligonucleotides 038
3.6 Chemicals, enzymes and other materials 040
3.7 Instruments 041
4 Methods 042
4.1 Molecular biology techniques 042
4.1.1 Restriction analysis 042
4.1.2 Polymerase Chain Reaction (PCR) 042
ITABLE OF CONTENTS
4.1.3 Cloning of PCR products 043
4.1.4 Agarose gel electrophoresis 043
4.1.5 Isolation of DNA from agarose gels 043
4.1.6 DNA quantification 043
4.1.7 Klenow reaction 043
4.1.8 T4 DNA polymerase reaction 044
4.1.9 Treatment with Calf Intestinal Alkaline Phosphatase (CIAP) 044
4.1.10 Ligation 044
4.1.11 Phenol/Chloroform extraction 044
4.1.12 E. coli colony PCR 044
4.1.13 DNA precipitation 044
4.1.14 Competent cells 045
4.1.15 Transformation of E. coli 045
4.1.16 Isolation of plasmid DNA from E. coli 046
4.1.17 DNA-sequencing 047
4.1.18 Site directed mutagenesis 047
4.2 Cellular biology techniques 048
4.2.1 Yeast transformation 048
4.2.2 Isolation of genomic DNA from S. cerevisiae 048
4.2.3 Yeast colony PCR 048
4.2.4 Mating 049
4.2.5 Sporulation and tetrad dissection 049
4.2.6 Cell synchronisation 049
4.2.7 Depletion of NDC80-Expression 050
4.2.8 Mps1-overexpression 050
4.2.9 Nocodazole survival assay 050
4.2.10 Quantification of Pds1-levels 051
4.2.11 Dot spots for growth analysis 051
4.2.12 Microscopy 051
4.2.13 FACS analysis 052
4.2.14 Chromatin immunoprecipitation - ChIP 052
IITABLE OF CONTENTS
4.2.15 Epitopal tagging of genes 054
4.2.16 CEN5-GFP tagging 054
4.2.17 Recycling of HIS3-marker 054
4.2.18 Yeast glycerol stocks 054
4.2.19 URA3/5’-FOA system 055
4.2.20 A system for the integration of an ndc80-mutant into the endogenous DNA-locus 055
4.2.21 Expression and purification of HIS -tagged proteins from E. coli 05710
4.3 Biochemical techniques 058
4.3.1 Yeast protein extracts 058
4.3.2 Bradford 058
4.3.3 SDS-PAGE 058
4.3.4 Coomassie staining 059
4.3.5 Western blotting 059
4.3.6 TAP purification 060
4.3.7 TCA protein precipitation 061
4.3.8 Kinase Assays 061
4.3.9 Sample preparation for the identification of phospho-sites by mass spectrometry 062
4.3.10 in vitro binding assay 062
5 Results 063
5.1 General characterisation of the Ndc80-complex 064
5.1.1 TAP-purification of the Ndc80-complex 064
5.1.2 Kinetochore localisation of the Ndc80-complex 064
5.2 Interaction between the Ndc80-complex and Mps1 067
5.2.1 Mps1 physically interacts with the Ndc80-complex in vivo 067
5.2.2 The Mps1 kinase weakly associates with the S. cerevisiae kinetochore in vivo 067
5.3 Phosphorylation of Ndc80 by Mps1 070
5.3.1 Mps1 directly phosphorylates Ndc80 in vitro 070
1-257 5.3.2 Mps1 physically interacts with Ndc80 in vitro 073
5.3.3 Phosphorylation of Ndc80 depends on Mps1 in vivo 073
5.4 Identification of Ndc80-phosphorylation sites by mass spectrometry 074
5.4.1 Strategy 074
IIITABLE OF CONTENTS
5.4.2 Identification of Ndc80-phosphorylation sites by MALDI-TOF 078
5.4.3 Confirmation of Ndc80-phosphorylation sites by LC-ESI-Quadrupole-TOF 080
5.5 Functional analysis of Ndc80-phosphorylation 083
5.5.1 Analysis of ndc80 deletion mutants 083
5.5.2 Spindle checkpoint analysis of ndc80∆1-43 and ndc80∆1-116 086
11A5.5.3 The spindle checkpoint of ndc80 is impaired 087
11D 5.5.4 ndc80 positively regulates the spindle checkpoint 088
14D 5.5.5 The lethality of the ndc80 mutant depends on the spindle checkpoint 090
14D 5.6 Functionality of the ndc80 protein 093
14D5.7 Detailed analysis of the ndc80 phenotype 095
14D 5.7.1 A system for the analysis of the lethal ndc80 mutant 095
14D5.7.2 The ndc80 mutant causes cell cycle arrest in mitosis 097
14D5.7.3 The spindle assembly checkpoint is permanently activated by the ndc80 mutant 098
14D5.7.4 ndc80 cells arrest with short spindles and sister kinetochores under tension 100
5.8 Mps1 is required for spindle checkpoint activation also downstream of Ndc80 103
14A5.9 The checkpoint of the non-phosphorylatable ndc80 mutant is deficient 104
6 Discussion 106
6.1 Ndc80 physically interacts with Mps1 106
6.2 Mps1 phosphorylates Ndc80 in vitro and in vivo 107
6.3 Mutagenesis strategy 110
6.4 Functional analysis of Ndc80-phosphorylation 111
11A 6.4.1 Non-phosphorylatable Ndc80 (ndc80 ) causes a defective spindle checkpoint 111
6.4.2 Pseudo-phosphorylated Ndc80 activates the spindle checkpoint 112
14D6.5 The ndc80 protein is part of a functional kinetochore 115
6.6 Mps1 is required for spindle checkpoint activation also downstream of Ndc80 116
6.7 A second role of Ndc80-phosphorylation 117
6.8 A Model for spindle checkpoint regulation by Ndc80-phosphorylation 118
7 References 120
Acknowledgements
IVABBREVEATIONS
ABBREVIATIONS
A alanine
aa amino acid
ade adenine
Amp ampicillin
ATP adenosine triphosphate
bp base pair
CBP Calmoduline binding peptide
CEN centromere
CHIP chromatin immunoprecipitation
Chr chromosome
°C degree celsius
D aspartate
DMSO dimethyl sulfoxide
DNA deoxyribonucleic acid
dNTP deoxynucleosid 5´-triphosphate
DOC deoxycholate
DTT dithiothreitol
E. coli Escherichia coli
EDTA Ethylenediaminetetraacetic acid
EtOH ethanol
FACS Fluorescence activated cell sorting
FOA 5´-fluoroorotic acid
g gram
GFP Green Fluorescent Protein
h hour
his histidine
IP immunoprecipitation
IPTG isopropyl-beta-D-thiogalactopyranoside
k 1000
kb kilo base
kDa kilo Dalton
LB Luria-Bertani media
leu leucine
LiAc lithium acetate
lys lysine
M molar
MALDI Matrix Assisted Laser Desorption/Ionisation
mega base pairsMbp
VABBREVEATIONS
min minute
ml milliliter
mg milligram
µg microgramm
µl microliter
µM micromolar
MT microtubule
ng nanogram
NZ nocodazole
o/n over night
OD587 optical density at 587 nm
ON oligonucleotide
ORF open reading frame
p plasmid
PAGE polyacrylamide gel electrophoresis
PBS Phosphate Buffered Saline
PCR polymerase chain reaction
PEG Polyethylene glycol
PMSF phenylmetanosulfonylfluoride
ProA Protein A
PMSF phenylmethylsulphonyl fluoride
PVDF polyvinylidendifluoride
rpm rotations per min
RT room temperature
S serine
S. cerevisiae Saccharomyces cerevisiae
SDS sodium dodecyl sulfate
sec second
SPB spindle pole body
t time
T threonine
TAP tandem affinity purification
trp tryptophane
ts temperature sensitive
U unit
ura uracil
WB Western Blot
WT wild-type
Y yeast strain
VI