Identification and analysis of Dictyostelium discoideum microtubule associated proteins [Elektronische Ressource] / vorgelegt von Katrin Veronika Koch

Identification and analysis
of Dictyostelium discoideum
microtubule associated proteins


Dissertation
zur
Erlangung des Doktorgrades
der Naturwissenschaften
(Dr. rer. nat.)


dem
Fachbereich Biologie
der Philipps-Universität Marburg
vorgelegt von

Katrin Veronika Koch
aus München

Marburg/Lahn 2006
























Vom Fachbereich Biologie der Philipps-Universität Marburg als Dissertation
angenommen am: 05. September 2006


Tag der mündlichen Prüfung: 27. November 2006


Erstgutachter: Prof. Dr. Michael Bölker
Zweitgutachter: Prof. Dr. Ralph Gräf Erklärung

Ich versichere, dass ich meine Dissertation selbständig, ohne unerlaubte Hilfe
angefertigt und mich dabei keiner anderen als der von mir ausdrücklich bezeichneten
Quellen und Hilfen bedient habe.

Die Dissertation wurde in der jetzigen oder einer ähnlichen Form noch bei keiner
anderen Hochschule eingereicht und hat noch keinen sonstigen Prüfungszwecken
gedient.




Marburg, im Oktober 2006 Katrin Veronika Koch


















Die Untersuchungen zur vorliegenden Arbeit wurden von April 2003 bis August 2006
im Labor von Prof. Dr. Manfred Schliwa am Adolf-Butenandt-Institut für Zellbiologie
der Ludwig-Maximilians-Universität in München unter der Betreuung von Prof. Dr.
Ralph Gräf durchgeführt. Betreuer an der Philipps-Universität Marburg war Prof. Dr.
Michael Bölker.
Part of this work has been published:

Koch, K.V., Reinders, Y., Ho, T.H., Sickmann, A., Gräf, R., 2006. Identification and
isolation of Dictyostelium microtubule-associated protein interactors by tandem
affinity purification. European Journal of Cell Biology. 85 (9-10).

Talks:
Koch, K.V. 2005. Dictyostelium discoideum - a model organism suitable for studying
Microtubule associated Protein Interactors by Tandem Affinity Purification; At:
Department of Botany Seminar. University of Toronto, Toronto, Canada.

Koch, K.V. 2005. Identification of Dictyostelium microtubule associated protein
thinteractors by Tandem affinity purification; At: 5 Biomedical Center (BMC) Seminar
Series. LMU, München, Germany.

Meeting abstracts:
Koch, K.V., Reinders, Y., Sickmann, A., Gräf, R. 2006. Tandem Affinity Purification in
Dictyostelium: Search for Interactors of EB1 and DdCP224 at the Centrosome and
the Microtubule Plus-ends, pp. 119 Jahrestagung DGZ, Vol. 85S1. European Journal
of Cell Biology, TU Braunschweig.

Koch, K.V., Reinders, Y., Sickmann, A., Gräf, R. 2005a. Tandem Affinity Purification
in Dictyostelium: Search for Interactors of EB1 and DdCP224 at the Centrosome and
the Microtubule Plus-ends. The American Society for Cell Biology 45th Annual
Meeting, Vol. 16. Molecular Biology of the Cell Supplement, Moscone Center, San
Francisco.

Koch, K.V., Reinders, Y., Sickmann, A., Gräf, R. 2005c. Screen for interactors of
DdEB1 and DdCP224 at the centrosome and the microtubule tip in Dictyostelium
SFB 413 Munich Symposium on Cell Dynamics: From Molecular Structure to Cellular
Motility, München/Martinsried.
Sickmann, A., Gräf, R. 2005b. Screen for interactors of Dictyostelium
EMBO Workshop Centrosomes and Spindle Pole Bodies. EMBO, EMBL Heidelberg.

Koch, K.V., Wagner, Y., Hestermann, A., Rehberg, M., Sickmann, A., Gräf, R. 2005d.
Screening for interactors of Dictyostelium EB1 and the XMAP215 family member
DdCP224 at the microtubule tip and the centrosome, pp. 51-52 Jahrestagung DGZ,
Vol. 84S1. European Journal of Cell Biology, Heidelberg.
hberg, M., Sickmann, A., Gräf, R. 2004b. Dictyostelium EB1 and the XMAP215 family member
DdCP224 at the microtubule tip and the centrosome 6th young Scientists Meeting
"Cytoskeletal Dynamics", Heidelberg.

Koch, K.V., Hestermann, A., Rehberg, M., Gräf, R. 2004a. Screening for interactors
of Dictyostelium EB1 and the XMAP215 family member DdCP224 at the microtubule
tip and the centrosome, pp. 42 Jahrestagung DGZ, Vol. 83. European Journal of Cell
Biology, Berlin.
Contents
Table of Contents

Abbreviations
Summary
Zusammenfassung
I Introduction 1
1.1 Dictyostelium discoideum as a model organism 1
1.2 The microtubule cytoskeleton 3
1.3 The centrosome 4
1.4 Microtubule plus end protein complex 6
1.5 MAPs 7
1.5.1 EB1 7
1.5.2 XMAP215 proteins and their Dictyostelium
discoideum member DdCP224 9
1.6 Aims of this study 10
II Materials and Methods 11
1 Materials 11
1.1 Reagents 11
1.2 Antibodies
1.3 Enzymes 12
1.4 Antibiotics 12
1.5 Buffers and solutions 12
1.6 Software 14
1.7 Other materials 14
2 Organisms and microbiological methods 14
2.1 Organisms 14
2.1.1 Dictyostelium strains 14
2.1.2 Bacterial Strains 14
2.1.3 S. cerevisiae strains 15
2.2 Cultivation and preservation of organisms 15
2.2.1 Media and cultivation of D. discoideum 15
2.2.2 Media and Cultivation of E. coli 16
2.2.3 Yeast media and cultivation 16
3 Molecular biology methods 18 Contents
3.1 DNA cleavage with restriction enzymes 18
3.2 Agarose gel electrophoresis 18
3.3 DNA extraction from agarose gels 18
3.4 Determination of DNA concentration 18
3.5 Preparation of plasmid DNA 19
3.6 Polymerase chain reaction (PCR) 19
3.7 Reverse Transcription – PCR (RT-PCR) 19
3.8 Oligonucleotides 20
3.9 Dephosphorylation of DNA 21
3.10 Ligation of DNA into plasmid vectors 22
3.11 Preparation and transformation of chemically and electro-
competent E. coli cells 22
3.11.1 Preparation of electrocompetent cells 22
3.11.2 Electroporation 22
3.11.3 Preparation of chemically competent cells 23
3.11.4 Heat Shock transformation 23
3.11.5 Identification of transformed clones in E. coli 23
3.12 Transformation of S. cerevisiae 23
3.13 Yeast two-hybrid screening 24
3.14 Preparation of plasmid DNA from yeast 24
3.15 Preparation of chromosomal DNA from D. discoideum 25
3.16 Transformation and cloning of D. discoideum 26
3.17 Isolation of polyadenylated RNA from D. discoideum 27
3.18 Generation of Constructs 27
4 Biochemical and immunological methods 29
4.1 SDS-Polyacrylamide gel electrophoresis (PAGE) 29
4.2 Coomassie and silver staining 29
4.2.1 Coomassie staining 29
4.2.2 Colloidal Coomassie staining 30
4.2.3 Silver staining 30
4.3 TCA precipitation of proteins 30
4.4 Western blots and immunostaining 30
4.5 Determination of protein concentration 31
4.6 Purification of bacterially expressed, MBP-tagged proteins 32 Contents
4.7 Preparation of whole cell extracts, nuclei and centrosomes from
D. discoideum 32
4.8 Antigen preparation and immunizations 33
4.9 Covalent coupling of antibodies and purified proteins to NHS-
activated sepharose 33
4.10 Affinity purification of antisera 34
4.11 Tandem affinity purification of D. discoideum protein complexes 34
4.11.1 Protein electrophoresis and mass spectrometry 35
4.12 Immunoprecipitation 36
4.13 GST-Pulldown 37
4.14 Isolation of Dictyostelium centrosomes 38
5 Cell biological methods 39
5.1 Indirect immunofluorescence microscopy on whole D. discoideum
cells and isolated centrosomes 39
5.2 Confocal microscopy 41
III Results 42
1 Screening for interactors of DdEB1 and DdCP224 employing the
yeast two hybrid system 42
1.1 Test for autoactivation of baits 43
1.2 Selection of clones of interest 45
1.3 Sequence analysis of putative interactors 46
1.4 Cloning of putative interactors 51
1.5 Investigation of subcellular localization by GFP fusion 52
1.6 Attempted verification by co-immunoprecipitation and GST pulldown
54
2 Screening for interactors by Tandem Affinity Purification 55
2.1 Generation of a Dictyostelium discoideum TAP-tag expression
vector 57
2.2 Cloning of baits into TAP vector 57
2.3 Expression and cleavability of constructs 58
2.4 Localization of tagged proteins 60
2.5 Adaptation of purification to Dictyostelium discoideum 61
2.6 Enrichment of fusion proteins 62
2.7 Analysis of putative interactors 63 Contents
3 DdTACC1 67
3.1 Sequence Analysis 68
3.2 Creation of a GFP-TACC domain fusion protein 70
3.3 Co-immunoprecipitation with DdCP224 71
3.4 Purification of TACC domain by MBP fusion 72
3.5 Generation of polyclonal antibodies against TACC domain 72
3.6 Isolation of centrosomes 73
3.7 Deletion construct 74
IV Discussion 76
1 Yeast two hybrid screening 76
1.1 Five putative interactors identified 76
1.2 Putative DdEB1 interactors show no co-localization 77
1.3 Putative interactors of N-terminal part of DdCP224 reveal auto-
phagy protein 78
1.4 Conclusions on yeast two hybrid screening 78
2 A n