Collective motor dynamics in membrane transport in vitro
Shaklee, P.M.
Citation
Shaklee, P. M. (2009, November 11). Collective motor dynamics in membrane transport in vitro. Retrieved from https://hdl.handle.net/1887/14329
Version: Corrected Publisher’s Version
License: Licence agreement concerning inclusion of doctoral thesis in the Institutional Repository of the University of Leiden
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Collective motor dynamics in membrane transport in vitro
Paige M. Shaklee
Promotiecommissie
Promotores: Prof. dr. M. Dogterom Prof. dr. T. Schmidt
Referent: Prof. dr. C. Schmidt (Universit¨at G¨ottingen, DE) Overige leden: Prof. dr. M. Orrit
Dr. ir. J. van Noort Dr. ir. E. J. G. Peterman
(Vrije Universiteit Amsterdam)
Dr. C. Storm (Technische Universiteit Eindhoven) Prof. dr. J. M. van Ruitenbeek
ISBN 978-90-6464-372-9
2009 Paige M. Shaklee. All rights reserved.c
The work described in this thesis was performed at the University of Leiden, Niels Bohrweg 2, 2333 CA, Leiden and the FOM-Institute for Atomic- and Molecular Physics, Science Park 113, 1098 XG, Amsterdam, The Netherlands.
This work is part of the research program of the “Stichting voor Fundamenteel Onderzoek der Materie (FOM)”, which is financially supported by the “Neder- landse organisatie voor Wetenschappelijk Onderzoek (NWO)” within the pro- gram on Material Properties of Biological Assemblies Grant FOM-L1708M.
Collective motor dynamics in membrane transport in vitro
PROEFSCHRIFT
ter verkrijging van de
graad Doctor aan de Universiteit Leiden, op gezag van Rector Magnificus
prof. mr. P.F. van der Heijden,
volgens besluit van het College voor Promoties te verdedigen op Woensdag 11 November 2009
klokke 13.45 uur
door
Paige Marie Shaklee
geboren te Stamford, CT, Verenigde Staten in 1981
Publications
This thesis is partly based on the following articles:
Paige M. Shaklee, Thomas Schmidt and Marileen Dogterom. Collec- tive motor dynamics in cargo transport. Review in preparation
(chapter 1)
Paige M. Shaklee∗, Stefan Semrau∗, Maurits Malkus, Stefan Kubick, Marileen Dogterom and Thomas Schmidt. Protein incorporation in giant lipid vesicles under physiological conditions. submitted
(chapter 2)
Paige M. Shaklee∗, Timon Idema∗, Gerbrand Koster, Cornelis Storm, Thomas Schmidt and Marileen Dogterom. 2008. Bidirectional motility of membrane tubes formed by nonprocessive motors. Proc. Natl. Acad.
Sci. USA 105:7993-7997.
(chapter 4)
Paige M. Shaklee, Line Bourel-Bonnet, Marileen Dogterom and Thomas Schmidt. Nonprocessive motor dynamics at the microtubule membrane tube interface. Biophys. J. accepted.
(chapter 5)
Paige M. Shaklee, Timon Idema, Line Bourel-Bonnet, Marileen Dogterom and Thomas Schmidt. Kinesin recycling in stationary membrane tubes. submitted
(chapter 6)
Contents
1 Introduction 9
1.1 Motor-driven transport . . . 10
1.2 Single motor studies . . . 10
1.3 From the individual to the collective . . . 12
1.4 Collective dynamics in membrane transport and tube pulling 15 1.5 Contents of the thesis . . . 18
2 Materials and Methods 21 2.1 Materials: vesicles, motors and microtubules . . . 22
2.1.1 Vesicle formation . . . 22
2.1.2 Microtubules . . . 27
2.1.3 Motor Proteins . . . 27
2.2 Experimental Assays . . . 31
2.2.1 Tube-pulling assay . . . 31
2.2.2 SUV transport assay . . . 34
2.3 Image Acquisition . . . 34
3 Image Correlation Spectroscopy and Fluorescence Recov- ery after Photobleaching in 1-D 37 3.1 Image Correlation Spectroscopy, 1-D . . . 38
3.1.1 Solution for the diffusion equation: single-species 1-D diffusion . . . 41
3.1.2 The Autocorrelation profile: single-species 1-D dif- fusion . . . 42
3.1.3 The Autocorrelation profile: 1-D diffusion with an additional directed motion . . . 43
3.2 Fluorescence Recovery After Photobleaching, 1-D . . . . 44 5
6 CONTENTS
3.2.1 FRAP: Simple 1-D diffusion . . . 44
3.2.2 FRAP: 1-D diffusion at the tip of a membrane tube 46 4 Bidirectional membrane tubes driven by nonprocessive motors 49 4.1 Membrane tubes formed by nonprocessive motors . . . . 51
4.2 Results: nonprocessive motors move membrane tubes bidi- rectionally . . . 53
4.2.1 Experimental results . . . 53
4.2.2 Model . . . 56
4.3 Discussion . . . 59
4.3.1 Simulations . . . 61
4.3.2 Conclusion . . . 63
4.4 Data Analysis . . . 64
5 Nonprocessive motor dynamics at the microtubule mem- brane tube interface 71 5.1 Nonprocessive motors in membrane tubes . . . 73
5.2 Results . . . 74
5.3 Fluorescence image correlation analysis . . . 77
5.4 Fluorescence recovery analysis . . . 84
5.5 Nature of the slowly diffusing fraction . . . 88
5.6 Data Analysis: FRAP . . . 89
6 Kinesin recycling in stationary membrane tubes 91 6.1 Processive motors in non-moving membrane tubes . . . . 92
6.2 Experimental results: kinesins cluster towards the tip at typical timescales . . . 93
6.3 Model and Simulations: cooperative binding, unbinding and a nucleation point . . . 98
6.4 Conclusion . . . 108
7 Bidirectional transport by competing kinesin and dynein, preliminary results 109 7.1 Models for bidirectional transport . . . 110
7.2 Tug-of-war . . . 112
7.3 Comparison of simulations to experimental data . . . 114
CONTENTS 7 7.4 Outlook . . . 121 7.5 Data Analysis . . . 122
Bibliography 125
Summary 139
Samenvatting 145
Curriculum Vitae 151
8 CONTENTS
