Microtubule associated molecular motors are involved in a multitude of fundamental cellular processes such as intracellular transport and spindle positioning. During these movements multiple motor proteins often work together and are, therefore, able to exert high forces. Thus force generation and sensing are common mechanisms for controlling motor driven movement. These mechanisms play a pivotal role when motor proteins antagonize each other, e.g. to facilitate oscillations of the spindle or the nucleus.
Single motor proteins have been characterized in depth over the last two decades, our understanding of the collective behavior of molecular motors remains, however, poor. Since motor proteins often cooperate while they walk along microtubules, it is necessary to describe their collective reaction to a load quantitatively in order to understand the mechanism of many motor-driven processes.
I studied the antagonistic action of many molecular motors (of one kind) in a gliding geometry. For this purpose I crosslinked two microtubules in an antiparallel fashion, so that they formed \"doublets\". Then I observed the gliding motility of these antiparallel doublets and analyzed the gliding velocity with respect to the relative number of motors pulling or pushing against each other. I observed that the antiparallel doublets gliding on conventional kinesin-1 (from Drosophila melanogaster) as well as cytoplasmic dynein (from Saccharomyces cerevisae) exhibited two distinct modes of movement, slow and fast, which were well separated. Furthermore I found a bistability, meaning, that both kinds of movement, slow and fast, occurred at the same ratio of antagonizing motors. Antiparallel doublets gliding on the non-processive motor protein Ncd (the kinesin-14 from D. melanogaster) showed, however, no bistability. The collective dynamics of all three motor proteins were described with a quantitative theory based on single-motor properties.
Furthermore the response of multiple dynein motors towards an external, well-defined load was measured in a gliding geometry by magnetic tweezing. Examples of multi-motor force-velocity relationships are presented and discussed. I established, furthermore, a method for counting single surface immobilized motors to guide the evaluation of the tweezing experiments.:1 Introduction to the functions of molecular motors 1
1.1 How molecular motors move 1
1.1.1 Of muscles and molecules 1
1.1.2 Kinesin-1, the working horse of single-molecule research 3
1.1.3 Kinesin-14, an unusual kinesin with a new twist 6
1.1.4 Cytoplasmic dynein, the molecule with many qualities 7
1.2 Structure and function of microtubules 8
1.3 The directionality of molecular motors 9
1.4 Force regulation in cell biology via molecular motors 10
1.4.1 Bidirectional cargo transport 10
1.4.2 Dynein drives intracellular oscillations 13
1.4.3 Control of spindle length 15
2 Introduction to the collective behavior of molecular motors in vitro 19
2.1 Cooperativity of molecular motors 19
2.2 How multiple motors work against a load 21
2.2.1 Theoretical concepts 21
2.2.2 Optical tweezing of multiple motors 22
2.2.3 Alternative experimental approaches 23
2.2.4 Membrane tube dynamics 24
2.3 Antagonizing molecular motors 25
2.3.1 Competition between dissimilar motors 25
2.3.2 Competition between identical motors 26
2.4 Aim of the project 28
3 Characterization of molecular motors 31
3.1 Results: The run length of processive motors 31
3.1.1 Run length of kinesin-1 at different ATP concentrations 31
3.1.2 The run length of cytoplasmic dynein 34
3.2 Results for multi-motor gliding assays 37
3.2.1 The effect of ATP on the gliding motility 37
3.2.2 The effect of temperature on the gliding motility 39
3.2.3 Bead transport does not influence gliding motility 42
3.3 Discussion 43
4 Magnetic tweezing of multiple molecular motors 45
4.1 Concepts of the magnetic tweezing setup 45
4.1.1 Theoretical concepts 45
4.1.2 Implementation 48
4.1.3 Calibration 51
4.2 Results of multi-motor force measurements 53
4.2.1 External force leads to microtubule re-orientation 53
4.2.2 Cytoplasmic dynein is able to withstand high opposing loads 55
4.2.3 Force-velocity curves at very low motor densities 56
4.2.4 Averaging of multi-motor force-velocity relationships 58
4.3 Discussion 60
5 Reconstitution of antagonizing motor activity 63
5.1 The doublet assay 63
5.2 Experimental results of the doublet assay 65
5.2.1 Kinesin-1 driven doublets move in discrete velocity regimes 65
5.2.2 Velocity affects the shape of the bistability curve 68
5.2.3 Dynein\'s processivity allows bistability at low velocity 69
5.2.4 Ncd does not exhibit a bistability curve 70
5.3 Theoretical results of the doublets assay 71
5.3.1 General concepts 71
5.3.2 Theory for processive motors 73
5.3.3 Theory for non-processive motors 75
5.3.4 The emergence of bistability 78
5.3.5 Model for single-motor force-velocity relationships 81
5.4 Comparison between theoretical and experiment results 83
5.5 Discussion 87
6 Materials and Methods 91
6.1 List of chemicals and equipment 91
6.2 Buffer recipes 92
6.3 Protein purification 93
6.4 Preparation of microtubules 95
6.5 Preparation of flow cells 96
6.6 Fluorescence microscopy 98
6.7 Errors computation 100
6.8 Software 100
7 References 103
8 Acknowledgement 113
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa:de:qucosa:25966 |
| Date | 23 March 2012 |
| Creators | Neetz, Manuel |
| Contributors | Tolic-Norrelykke, Iva, Schwille, Petra, Gennerich, Arne, Technische Universität Dresden |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | English |
| Detected Language | English |
| Type | doc-type:doctoralThesis, info:eu-repo/semantics/doctoralThesis, doc-type:Text |
| Rights | info:eu-repo/semantics/openAccess |
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