Functional organization of the actin system in Dictyostelium [Elektronische Ressource] / Hellen Cristina Ishikawa-Ankerhold

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Sujets

• Biologie

Informations

• Publié par: technische_universitat_munchen
• Publié le: 01 janvier 2009
• Nombre de lectures: 26
• Langue: Deutsch
• Poids de l'ouvrage: 5 Mo

Extrait

TECHNISCHE UNIVERSITÄT MÜNCHEN

Max Planck Institute of Biochemistry


Functional Organization of the
Actin System in Dictyostelium



Hellen Cristina Ishikawa-Ankerhold



Vollständiger Abdruck der von der Fakultät für Chemie der
Technischen Universität München zur Erlangung des akademischen
Grades eines Doktors der Naturwissenschaften genehmigten
Dissertation.




Vorsitzender: Univ.-Prof. Dr. J. Buchner
Prüfer der Dissertation:
1. Hon.-Prof. Dr. W. Baumeister
2. Univ.-Prof. Dr. S. Weinkauf

Die Dissertation wurde am 02.12.2008 bei der Technischen Universität München
eingereicht und durch die Fakultät für Chemie am 08.04.2009 angenommen.

Table of Contents
1 Introduction................................................................................1
1.1 Dictyostelium discoideum as a model to study actin dynamics................................1
1.2 Cytokinesis...............................................................................................................3
1.3 The centrosome .......................................................................................................4
1.4 Phagocytosis in D. discoideum5
1.5 Chemotaxis ..............................................................................................................8
1.6 Function of cAMP...................................................................................................10
1.7 The actin cytoskeleton ...........................................................................................11
1.8 Actin-associated proteins in Dictyostelium discoideum..........................................12
1.8.1 The Arp2/3 complex ...................................................................................14
1.8.2 WASP and neural (N)-WASP.....................................................................16
1.8.3 The SCAR complex....................................................................................17
1.8.4 Coronin.......................................................................................................19
1.8.5 Actin-interacting protein 1 ..........................................................................26
1.8.6 LimE protein ...............................................................................................28
1.9 Oscillations in cell biology ......................................................................................29
1.9.1 Oscillations of cytoskeletal structures ........................................................30
1.10 Microfluidic device..................................................................................................31
1.11 Caged compounds.................................................................................................32
1.12 Aim of this study.....................................................................................................33
2 Materials and Methods............................................................34
2.1 Materials.................................................................................................................34
2.1.1 Antibodies34
2.1.2 Dyes and others .........................................................................................34
2.1.3 Antibiotics...................................................................................................34
2.1.4 Buffers and solutions..................................................................................34
2.1.5 Agar overlay ...............................................................................................35
2.2 Cell Biological Methods..........................................................................................37
2.2.1 Cell Culture ................................................................................................37
2.2.2 Preservation of Dictyostelium discoideum .................................................38
2.2.3 Transformation and electroporation of Dictyostelium cells.........................38
2.2.4 Cell lines transformed ................................................................................38
2.2.5 Other cell lines transformed .......................................................................39
I
2.2.6 Cell isolation via Laser Microdissection and Pressure Catapulting............40
2.2.7 Microfluidic device......................................................................................41
2.2.8 Fluorescence microscopy and photo-activation .........................................42
2.2.9 Image processing.......................................................................................43
2.2.10 Total Internal Reflection Fluorescence Microscopy................................44
2.2.11 Micropipette assay .................................................................................44
2.2.12 Phagocytosis assay................................................................................45
2.2.13 Cell velocity assay..................................................................................45
2.2.14 Immunofluorescence labeling.................................................................45
2.3 Biochemical Methods and Immunoblotting ............................................................46
2.3.1 SDS-Polyacrylamide gel electrophoresis ..................................................46
2.3.2 Coomassie blue staining ............................................................................46
2.3.3 Western blots and immunostaining47
2.3.4 Estimation of the G-/F-actin ratio ...............................................................47
2.3.5 Quantitative F-actin assay..........................................................................48
3 Results
Actin-filament turnover mediated by coronin and Aip1 is required for proper
cytokinesis, phagocytosis and motility in D. discoideum ...............................49
3.1 CorA/Aip1-null mutants have high filamentous actin content.................................50
3.2 Aggregation of D. discoideum cells is delayed in the absence of CorA and Aip1..53
3.3 CorA and Aip1 contribute to chromosome segregation and cytokinesis................54
3.4 The phagocytosis process is prolonged in the absence of CorA and Aip1 ............56
3.5 CorA/Aip1 double-mutant cells migrate slowly but still orient towards a cAMP
gradient ..................................................................................................................58
4 Results
Well-defined chemoattractant stimuli modulate protein recruitment to the cell
cortex of D. discoideum......................................................................................61
4.1 The temporal pattern of cAMP stimulation affects CorA and Aip1 translocation to
the cell cortex .........................................................................................................63
4.2 The rates of actin assembly and disassembly are affected in the absence of CorA
and Aip1 .................................................................................................................66
4.3 Pulses of cAMP promote resonant actin responses with periods of 20 or 40
seconds..................................................................................................................71
4.4 Resonant actin response is promoted in the presence of CorA and Aip1, but not in
their absence..........................................................................................................75
Ishikawa-Ankerhold II
5 Results
Cell-autonomous oscillations of actin polymerization in the cortex of SCAR-
deficient cells.......................................................................................................80
5.1 Autonomous and periodic fluctuations of actin polymerization in the cortex of
SCAR-deficient cells..........................................................................................................81
5.2 The developmental stage of SCAR-deficient cells has no influence on autonomous
fluctuations of actin polymerization in the cell cortex.........................................................85
5.3 Development of D. discoideum appears not to be affected by the absence of SCAR
and PIR121........................................................................................................................87
5.4 Actin redistribution at the basal cell cortex exhibits different patterns in the absence
of SCAR or PIR121............................................................................................................88
5.5 Localization of the Arp2/3 complex is not disturbed in the absence of SCAR .......92
5.6 CorA is recruited to the leading edge in the absence of SCAR .............................94
5.7 External cAMP stimulation promotes a shift in the phase of actin oscillations in
SCAR and PIR121-deficient cells......................................................................................95
5.8 The Arp2/3 complex is poorly recruited to the leading edge in the absence of
SCAR in a gradient of cAMP .............................................................................................99
6 Discussion ..........................