(Downloads - 0)
For more info about our services contact : help@bestpfe.com
Table of contents
1 Introduction
1.1 Cell cycle and cell division
1.1.1 The cell cycle
1.1.2 Cell division
1.2 The mitotic bipolar spindle
1.2.1 Microtubules
1.2.2 The microtubule associated proteins (MAPs)
1.2.3 The molecular motors
1.2.3.1 The dynein superfamily
1.2.3.1.1 Classification and structure of dyneins
1.2.3.1.2 Motor properties of cytoplasmic dynein
1.2.3.1.3 Function and regulation of cytoplasmic dynein
1.2.3.2 The kinesin superfamily
1.2.3.2.1 Classification and structure of kinesins
1.2.3.2.2 Motor properties of kinesins
1.2.3.2.3 Function and regulation of kinesins
1.2.4 Spindle architecture
1.3 The Xenopus kinesin-5: XlEg5
1.3.1 Eg5 homologs
1.3.2 Structure of Eg5
1.3.3 Motor properties of Eg5 in vitro
1.3.4 Function of Eg5 in vivo
1.3.4.1 Eg5 function in bipolar spindle formation and maintenance
1.3.4.2 Eg5 function in microtubule poleward flux
1.3.4.3 Eg5 function in a potential spindle matrix
1.3.5 Regulation of Eg5
1.3.5.1 Cdk1 phosphorylation of Eg5
1.3.5.2 Eg2 phosphorylation of Eg5
1.3.5.3 RanGTP regulation of Eg5
2 Motivation
3 Results
3.1 Role of Eg5 phosphorylation in bipolar spindle formation in Xenopus egg extract
3.1.1 Eg2 phosphorylates full length Eg5 at serine 543
3.1.1.1 Eg2 phosphorylates truncated GST-Eg5 at serine 543 in vitro and in mitotic egg extract
3.1.1.2 Eg2 phosphorylates full length Eg5 on Serine 543 in vitro
3.1.2 Cdk1 is the major kinase that phosphorylates Eg5 in Xenopus egg extract .
3.1.3 Eg5 S543A’ and Eg5 T937A are functional motors in vitro
3.1.4 Cdk1 phosphorylation, but not Eg2 phosphorylation is required for spindle assembly in Xenopus egg extract
3.1.5 Eg5 T937A localization to spindle microtubule is disrupted in Xenopus egg extract
3.2 Role of Eg5 intrinsic motor properties in spindle formation in Xenopus egg extract.
3.2.1 Eg5 chimeras
3.2.1.1 Kid-Eg5, Dkhc-Eg5 support microtubule gliding
3.2.1.2 The specific properties of Eg5 motor domain are required for spindle formation in Xenopus egg extract
3.2.2 Eg5 motility in mitotic Xenopus egg extract, development of an assay
3.2.2.1 Microtubule gliding assay in mitotic Xenopus egg extract
3.2.2.2 Eg5 motility in mitotic Xenopus egg extract
4 Discussion
4.1 Eg5 phosphorylation by Eg2 is not required for bipolar spindle formation in Xenopus egg extract.
4.2 Cdk1 phosphorylation regulates Eg5 efficient binding to spindle microtubule
4.3 Eg5 intrinsic motor properties are required for spindle assembly in Xenopus egg extract
4.4 Eg5 motility in Xenopus egg extract
5 Material and methods
5.1 Cloning
5.1.1 Full length Eg5 constructs
5.1.2 Chimeric Eg5 constructs
5.2 Proteins expression and purification
5.2.1 Expression and purification of Eg5 constructs
5.2.2 Expression and purification of Eg2
5.2.3 Expression and purification of p50
5.2.4 Antibody purification
5.2.4.1 Eg5 antibody
5.2.4.2 XKCM1 antibody
5.2.4.3 Katanin antibody
5.3 Phosphorylation experiments
5.4 Xenopus egg extract experiment
5.4.1 Preparation of Xenopus egg extract
5.4.2 Depletion-add back experiments
5.5 Microtubule gliding assay
5.5.1 In vitro microtubule gliding assay
5.5.2 In extract microtubule gliding assay
6 References
7 Appendix
7.1 Résumé
7.2 Abstract
7.3 Abbreviations
7.4 Publications
