Collective motor dynamics in membrane transport in vitro. Paige M. Shaklee

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1 Collective motor dynamics in membrane transport in vitro Paige M. Shaklee

2 Promotiecommissie Promotores: Referent: Overige leden: Prof. dr. M. Dogterom Prof. dr. T. Schmidt Prof. dr. C. Schmidt (Universität Göttingen, DE) 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 c 2009 Paige M. Shaklee. All rights reserved. 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 Nederlandse organisatie voor Wetenschappelijk Onderzoek (NWO) within the program on Material Properties of Biological Assemblies Grant FOM-L1708M.

3 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 uur door Paige Marie Shaklee geboren te Stamford, CT, Verenigde Staten in 1981

4 Publications This thesis is partly based on the following articles: Paige M. Shaklee, Thomas Schmidt and Marileen Dogterom. Collective 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 Bidirectional motility of membrane tubes formed by nonprocessive motors. Proc. Natl. Acad. Sci. USA 105: (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)

5 Contents 1 Introduction Motor-driventransport Singlemotorstudies From the individual to the collective Collective dynamics in membrane transport and tube pulling Contents of the thesis Materials and Methods Materials: vesicles, motors and microtubules Vesicle formation Microtubules Motor Proteins ExperimentalAssays Tube-pulling assay SUVtransportassay Image Acquisition Image Correlation Spectroscopy and Fluorescence Recovery after Photobleaching in 1-D ImageCorrelationSpectroscopy,1-D Solution for the diffusion equation: single-species 1-Ddiffusion The Autocorrelation profile: single-species 1-D diffusion The Autocorrelation profile: 1-D diffusion with an additional directed motion Fluorescence Recovery After Photobleaching, 1-D

6 6 CONTENTS FRAP:Simple1-Ddiffusion FRAP: 1-D diffusion at the tip of a membrane tube 46 4 Bidirectional membrane tubes driven by nonprocessive motors Membrane tubes formed by nonprocessive motors Results: nonprocessive motors move membrane tubes bidirectionally Experimentalresults Model Discussion Simulations Conclusion DataAnalysis Nonprocessive motor dynamics at the microtubule membrane tube interface Nonprocessive motors in membrane tubes Results Fluorescence image correlation analysis Fluorescence recovery analysis Nature of the slowly diffusing fraction Data Analysis: FRAP Kinesin recycling in stationary membrane tubes Processive motors in non-moving membrane tubes Experimental results: kinesins cluster towards the tip at typical timescales Model and Simulations: cooperative binding, unbinding and a nucleation point Conclusion Bidirectional transport by competing kinesin and dynein, preliminary results Modelsforbidirectionaltransport Tug-of-war Comparison of simulations to experimental data

7 CONTENTS Outlook DataAnalysis Bibliography 125 Summary 139 Samenvatting 145 Curriculum Vitae 151

8 8 CONTENTS