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Kinesin motor stepping challenged
Kushwaha, V.S.
2018
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Kushwaha, V. S. (2018). Kinesin motor stepping challenged: The impact of traffic jams and temperature.
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KINESIN MOTOR STEPPING CHALLENGED
THE IMPACT OF TRAFFIC JAMS AND TEMPERATURE
I would like to acknowledge the following members of the reading committee for reviewing my thesis:
prof. dr. G.J.L. Wuite, Vrije Universiteit Amsterdam, The Netherlands prof. dr. M.L. Groot, Vrije Universiteit Amsterdam, The Netherlands prof. dr. ir. G. Woehlke, Technical University of Munich, Germany prof. dr. W.H. Roos, University of Groningen, The Netherlands
prof. dr. C. Storm, Technical University of Eindhoven, The Netherlands
This work is supported by the research grant ‘Barriers in the Brain’ of the Foundation for Fundamental Research on Matter (FOM) now known as NWO-I, which is part of the Netherlands Organisation for Scientific Research (NWO). All rights are reserved. No part(s) of this thesis shall be printed, reproduced or transmitted in any form without prior permission from the author.
© 2018, Vandana Singh Kushwaha ISBN: 978-94-9301-410-7
Printed by Gildeprint
VRIJE UNIVERSITEIT
KINESIN MOTOR STEPPING CHALLENGED
THE IMPACT OF TRAFFIC JAMS AND TEMPERATURE
ACADEMISCH PROEFSCHRIFT
ter verkrijging van de graad Doctor
aan de Vrije Universiteit Amsterdam,
op gezag van de rector magnificus
prof. dr. V. Subramaniam,
in het openbaar te verdedigen
ten overstaan van de promotiecommissie
van de Faculteit der Bètawetenschappen
op dinsdag 25 september 2018 om 11.45 uur
in de aula van de universiteit,
De Boelelaan 1105
door
Vandana Singh Kushwaha
“One does not discover new lands without consenting to lose sight of the shore for a very long time”
CONTENTS
Chapter 1: Introduction ... 1
1.1 Intracellular transport ... 1
1.2 Microtubules ... 2
1.3 Biological molecular motors ... 4
1.3.1 Kinesin-1: A conventional kinesin motor protein from Kinesin-1 family ... 5
1.3.2 Kinesin-2 family ... 9
1.4 Total internal reflection fluorescence microscopy ... 11
1.5 Single-motor motility assays ... 12
1.6 Outline of this thesis ... 13
References ... 15
Chapter 2: Crowding dynamics of Kinesin-1 and OSM-3 motor proteins 21 2.1 Introduction ... 22
2.2 Correlation imaging technique ... 24
2.3 Testing and extending the TASEP-LK model ... 32
2.4 Discussion and conclusions ... 37
Appendix A: Experimental methods ... 39
Appendix B: Correlation imaging ... 44
Appendix C: Particle tracking And TASEP ... 50
Supplementary materials ... 52
References ... 61
3.2 Materials and methods ... 67
3.2.1 Microtubules preparation ... 67
3.2.2 Cloning and preparation of motor proteins ... 68
3.2.3 Kinesin-II-microtubule crowding assay ... 69
3.2.4 Image acquisition and data analysis ... 70
3.3 Results ... 73
3.3.1 Probing the motility properties of purified Kinesin-II at the single-molecule level ... 73
3.3.2 Determination of Kinesin-II motility properties under crowded conditions using correlation-based image analysis ... 75
3.3.3 Kinesin-II density increases linearly with concentration, up to high concentrations ... 78
3.3.4 Kinesin-II velocity decreases only at very high density ... 79
3.3.5 Run length of Kinesin-II motors changes with density ... 80
3.3.6 TASEP-LK modelling ... 81
3.4 Discussion ... 82
Supplementary materials ... 86
References ... 87
Chapter 4: The temperature dependence of kinesin motor-protein mechanochemistry ... 91
4.1 Introduction ... 92
4.2 Materials and methods ... 94
4.2.1 Microtubules preparation ... 94
4.2.2 Cloning and preparation of motor proteins ... 94
4.2.3 Instrumentation ... 94
4.2.4 In vitro single-molecule motility assays ... 95
4.2.5 Temperature control ... 96
4.2.6 Single-particle tracking and data analysis ... 98
4.3.1 Kinesin motility parameters at different temperatures ... 98
4.3.2 The temperature dependence of kinesin motility parameters can be well described by a minimal 2-state model ... 101
