Exploring the regulation and function of the human guanine nucleotide exchange factor Ect2 (epithelial cell transforming protein 2) in cytokinesis [Elektronische Ressource] / vorgelegt von Ravindra B. Chalamalasetty

Ravindra Babu Chalamalasetty

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2006
• Nombre de lectures: 15
• Langue: Deutsch
• Poids de l'ouvrage: 2 Mo

Extrait

Exploring the regulation and function of the human
guanine nucleotide exchange factor Ect2 (epithelial cell
transforming protein 2) in cytokinesis




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



Vorgelegt von
Ravindra B. Chalamalasetty

Martinsried / München 2006
















Dissertation eingereicht am 02, February, 2006
Tag der mündlichen Prüfung : 08, March, 2006


Erstgutachter: Prof. Dr. Erich A. Nigg
Zweitgutachter: Prof.Dr. Stefan Jentsch




Hiermit erkläre ich, dass ich die vorliegende Dissertation selbständig und ohne unerlaubte
Hilfe angefertigt habe. Sämtliche Experimente sind von mir selbst durchgeführt worden,
falls nicht explizit auf dritte verwiesen wird. Ich versichere, daß ich weder versucht habe,
eine Dissertation oder Teile einer Dissertation an einer anderen Stelle einzureichen, noch
eine Doktorprüfung durchzuführen.

München, den 24-01-2006



Table of contents

Table of contents

TABLE OF CONTENTS…………………………………………...….I-IV

ACKNOWLEDGEMENTS

SUMMARY………………………………………………………………..1

1.0 INTRODUCTION…………………………………………………….3
1.1 Overview of the cell cycle……………………………………………..3
1.1.1 An overview of mitosis………………………………………………………..4
1.1.2 An overview of cytokinesis…………………………………………………....6
1.1.3 Regulation of M phase progression…………………………………………....6

1.2 Cytokinesis…………………………………………………………….10
1.2.1 Cytokinesis in yeasts and plants……………………………………………...10

1.3 Cytokinesis in mammalian cells……………………………………...14
1.3.1 Division site determination in mammalian cells…………………………......14
1.3.2 Models for the roles of microtubules in cleavage furrow formation……........15

1.4 Central spindle and contractile ring formation…………………….20
1.4.1 The central spindle and its components…………………………...............…20
1.4.2 Cleavage furrow determination…………………………….......................…21
1.4.3 The contractile ring and formation of the cleavage furrow …………………23

1.5 Ect2........................................................................................................27
1.5.1 Ect2, a Rho family GEF required for cytokinesis……………………….......27
ITable of contents
1.5.2 Other functions of Ect2………………………………………….............…28
1.5.3 Ect2 structure: different domains of Ect2………………………….........…29
1.5.4 Regulation of Ect2 during mitosis……………………………....................32

1.6 GOAL OF MY RESEARCH............................................................34

2.0 RESULTS..........................................................................................35
2.1 Mitotic phosphorylation of human Ect2.........................................35
2.1.1 Production of polyclonal Ect2 antibodies…………………………...........35
2.1.2 Ect2 is phosphorylated during early mitosis………………………...........37
2.1.3 Identification of multiple phosphorylation sites in mitotic Ect2…….........39
2.1.4 Plk1 can phosphorylate Ect2 in vitro…………………………………......42
2.1.5 No obvious interaction between Ect2 and Plk1 kinase……………….......46
2.1.6 Analysis of Ect2 phosphorylation site mutants……………………….......47
2.1.7 Conclusion…………………………...........................................................52

2.2 Regulation of Ect2 localization.........................................................53
2.2.1 Ect2 localizes predominantly to the central spindle and cell cortex….......53
2.2.2 The amino-terminal BRCT domain is required for central spindle
targeting, whereas the carboxyl-terminal PH domain targets Ect2 to the
cell cortex....................................................................................................54
2.2.3 Ect2 is targeted to the central spindle via the MKlp1/MgcRacGAP and
Aurora-B/MKlp2 complexes…………………………………….…….....56
2.2.4 Ect2 interacts with the MKlp1/MgcRacGAP complex via the amino-
terminal BRCT domain………………………………………………......60
2.2.5 The interaction between the BRCT domain of Ect2 and the MKlp1 /
MgcRacGAP might be phosphorylation dependent…………………......62
2.2.6 Ect2 central spindle localization is not essential for cytokinesis……......64
2.2.7 Conclusion………………………………………………….....................66

IITable of contents

2.3 Requirement of Ect2 in cytokinesis..................................................67
2.3.1 Ect2 controls both early and late cytokinesis events………........................67
2.3.2 RhoA and Citron kinase are not targeted to the cleavage furrow in
Ect2 depleted cells……………………........................................................70
2.3.3 Nuclear targeting of the amino-terminal Ect2 (1-333) fragment can prevent
cytokinesis defects…………………...........................................................73
2.3.4 The amino-terminal BRCT-containing fragment (1-333) is mislocalized
as a ring-like structure perpendicular to the midbody during
cytokinesis………………………………………………………………..76
2.3.5 Conclusion…………………………………………………………….......78

3.0 DISCUSSION....................................................................................79
3.1 Mitotic phosphorylation of Ect2……………………...................................79
3.2 Ect2 targeting and oncogenic potential………………………….................82
3.3 A model for Ect2 targeting to the central spindle………………………….83
3.4 Ect2 targets RhoA to the cleavage furrow independently of its central
spindle localization………..........................................................................85
3.5 Interference with Ect2 function blocks cytokinesis by impairing both
cleavage furrow formation and ingression………………...........................87

4.0 MATERIALS AND METHODS....................................................91
4.1 Materials………………………………………………………………......91
4.2 Plasmid constructions and site directed mutagenesis…..............................91
4.3 Antibodies………………………………………………….……………...93
4.4 Binding of antibodies to beads…………………………............................95
4.5 Generation of recombinant baculoviruses……………………….…..........96
4.6 Production of GST-Ect2 protein from sf9 insect cells…………................96
4.7 siRNA experiments……………………………………………….............97
4.8 Cell culture and generation of stable cell lines…………………...............98
4.9 Cell extracts, immunoprecipitations and western blot analysis………......98
IIITable of contents
4.10 Immunoprecipitation of endogenous Ect2 from HeLa S3 spinner
culture cells...............................................................................................100
4.11 Cell cycle profile and flow cytometry analysis…………………….…....101
4.12 Immunofluorescence microscopy....................................................….....101
4.13 In vitro kinase assays………………………………………………… ...102
4.14 Live-cell imaging......................................................................................103
4.15 Mass spectrometry…………………………………................................103

LIST OF ABBREVIATIONS...............................................................105

REFERENCES......................................................................................109

APPENDIX: LIST OF