Regulation of the actin cytoskeleton by Ste20-like kinases and the arginyl transferase 1 in Dictyostelium discoideum [Elektronische Ressource] / vorgelegt von Petros Batsios

Petros Batsios

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2009
• Nombre de lectures: 15
• Langue: English
• Poids de l'ouvrage: 5 Mo

Extrait

REGULATION OF THE ACTIN CYTOSKELETON BY
STE20-LIKE KINASES AND THE ARGINYL
TRANSFERASE 1 IN DICTYOSTELIUM DISCOIDEUM



Dissertation der Fakultät für Biologie der
Ludwig-Maximilian-Universität
München



vorgelegt von
PETROS BATSIOS
aus München

September, 2009









The experimental part of the work was carried out in the laboratory of Prof. Dr. Michael
Schleicher from October 2005 to March 2009 at the Institute for Cell Biology, Ludwig-
Maximilian-Universität München.

2Ehrenwörtliche Versicherung
Diese Dissertation wurde selbstständig und ohne unerlaubte Hilfsmittel angefertigt.


München, September 2009 Petros Batsios











1. Gutachter: Prof. Dr. Michael Schleicher
2. Gutachter: Prof. Dr. Angelika Boettger

Rigorosum am 22.01.2010
Papers:
2007
Rohlfs, M., Arasada, R., Batsios, P., Janzen, J., Schleicher, M. (2007). The Ste20-like
kinase SvkA of Dictyostelium discoideum is essential for late stages of cytokinesis. J Cell
Sci. 120:4345-4354.
Talks:
2008
Batsios, P. (2008). The Ste20-like kinase Krs2 is required for cell migration during
chemotaxis. 33rd FEBS Congress - 11th IUBMB conference. Athens, Greece. FEBS J, Vol.
275, Issue s1, Abstract: OP4D-1.
Meeting abstracts:
2008
Batsios, P., Schleicher, M., and Müller-Taubenberger, A. (2008). The role of arginylation
for actin dynamics in Dictyostelium discoideum. ASCB Annual meeting. San Francisco,
USA. Mol. Biol. Cell 19 (suppl), 369 B313.
2007
Batsios, P., Arasada, R., Schleicher, M., and Rohlfs, M. (2007). The STE20-like Severin
kinase of Dictyostelium discodeum is essential for late stages of cytokinesis. ASCB & ECF,
Summer meeting. Dijon, France. Abstract: 35.
2006
Batsios, P., Arasada, R., Rohlfs, M., und Schleicher, M. (2006). The STE20-like kinase
DST10 is involved in phagocytosis and late development of Dictyostelium discoideum.
Deutsche Gesellschaft für Zellbiologie, Jahrestagung. Braunschweig, Germany. Eur. J. Cell
Biol, 85S1 (supp. 56), O-23.
TABLE OF CONTENTS
SUMMARY............................................................................................................................1
ZUSAMMENFASSUNG .......................................................................................................3
1. Introduction.....................................................................................................................5
1.1 Ste20-like kinases ...................................................................................................5
1.2 Actin and actin regulation.....................................................................................10
1.3 Post-translational modifications of actin ..............................................................12
1.4 Dictyostelium discoideum as model organism......................................................15
1.5 Goals of the project...............................................................................................16
2. Materials and Methods..................................................................................................18
2.1 Materials ...............................................................................................................18
2.1.1 Computer programs ..........................................................................................19
2.1.2 Reagents............................................................................................................19
2.1.3 Media ................................................................................................................20
2.1.4 Buffers...............................................................................................................20
2.1.5 Instruments and centrifuges ..............................................................................20
2.1.6 Bacteria strains..................................................................................................22
2.1.7 D. discoideum strains........................................................................................22
2.1.8 Vectors ..............................................................................................................23
2.2 Methods.................................................................................................................24
2.2.1 Molecular Biological Methods ........................................................................24
2.2.2 Biochemical Methods .......................................................................................24
2.2.2.1 SDS-Polyacrylamide Gel Electrophoresis and Western blotting..............24
2.2.2.2 GST-tagged protein expression.................................................................25
2.2.2.3 Immunprecipitation and Nanotrap ............................................................26
2.2.2.4 Two-dimensional gel electrophoresis .......................................................26
2.2.3 Cell Biological Methods ...................................................................................27
2.2.3.1 D. discoideum growth in liquid medium and on agar plates.....................27
2.2.3.2 Analysis of cell shape and cell migration .................................................28
2.2.3.3 Chemotaxis ...............................................................................................28
2.2.3.4 Phagocytosis and pinocytosis assay..........................................................28
2.2.3.5 Phototaxis..................................................................................................29
2.2.3.6 Indirect immunofluorescence....................................................................29
I
2.2.3.7 Reflection Interference Contrast Microscopy...........................................30
2.2.3.8 Total Internal Reflection Fluorescence Microscopy.................................30
3. Results...........................................................................................................................31
3.1 Ste20-like kinases in D. discoideum .....................................................................31
3.1.1 The Ste20-like kinase DstA in D. discoideum..................................................31
3.1.1.1 DstA antibodies.........................................................................................34
3.1.1.2 Subcellular localization of DstA...............................................................37
3.1.1.3 Isolation of dst1 knockouts .......................................................................41
3.1.1.4 Phenotype characterization of the dst1 null..............................................42
3.1.1.5 Interactors for DstA ..................................................................................50
3.1.2 The Ste20-like kinase Krs2 in D. discoideum...................................................55
3.1.2.1 Sequence analysis of Krs2 ........................................................................55
3.1.2.2 Generation of Krs2 antibodies ..................................................................62
3.1.2.3 Subcellular localization of Krs2................................................................63
3.1.2.4 Isolation of krsB knockouts ......................................................................68
3.1.2.5 Characterization of the krsB null phenotype.............................................69
3.1.2.6 Interactors for Krs2...................................................................................75
3.2 Actin isoforms and actin modifications in D. discoideum....................................77
3.2.1 Actin isoforms in D. discoideum77
3.2.2 The Arg-tRNA transferase (Ate1) in D. discoideum ........................................79
3.2.2.1 Sequence analysis of Ate1 ........................................................................79
3.2.2.2 Subcellular localization.............................................................................83
3.2.2.3 Characterization of the ate1 null phenotype .............................................84
4. Discussion.....................................................................................................................91
4.1 The Ste20-like kinase DstA ..................................................................................91
4.2 The Ste20-like kinase Krs2...................................................................................94
4.3 Actin isoforms and post-translational modifications ............................................95
5. References98
List of Figures108
List of Tables ......................................................................................................................110
Acknowledgements.............................................................................................................111
Curriculum Vitae ................................................................................................................112

IISUMMARY
The regulation of actin by actin-binding proteins and salt conditions is very well studied.