Intracellular transport of membrane-bound vesicles and organelles is a process fundamental for many cellular functions including cell morphogenesis and signaling. The transport is mediated by ensembles of motor proteins, such as kinesins, walking on microtubule tracks. When transporting membrane-bound cargo inside a cell, the motors are linked to diffusive lipid bilayers either directly or via adaptor molecules. The fluidity of the lipid bilayers induces loose inter-motor coupling which is likely to impact the collective motor dynamics and may induce cooperativity. Here, we investigate the influence of loose coupling of kinesin motors on its transport characteristics.
In the first part of this thesis, we used truncated kinesin-1 motors with a streptavidin-binding-peptide (SBP) tag and performed gliding motility assays on streptavidin-loaded biotinylated supported lipid bilayers (SLBs), so called ‘membrane-anchored’ gliding motility assays. We show that the membrane-anchored motors act cooperatively; the microtubule gliding velocity increases with increasing motor density. This is in contrast to the transport behavior of multiple motors rigidly bound to a substrate. There, the motility is either insensitive to the motor density or shows negative interference at higher motor density, depending on the structure of the motors.
The cooperativity in transport driven by membrane-anchored motors can be explained as following: while stepping on a microtubule, membrane-anchored motors slip backwards in the viscous membrane, thus propelling the microtubule in the solution at a velocity, given by the difference of the motor stepping velocity and the slipping velocity. The motor stepping on the microtubule occurs at maximal stepping velocity because the load on the membrane-anchored motors is minute. Thus, the slipping velocity of membrane-anchored motors determines the microtubule gliding velocity. At steady state, the drag force on the microtubule in the solution is equal to the collective drag force on the membrane-anchored motors slipping in the viscous membrane. As a consequence, at low motor density, membrane-anchored motors slip back faster to balance the drag force of the microtubule in the solution. This results in a microtubule gliding velocity significantly lower than the maximal stepping velocity of the individual motors. In contrast, at high motor density, the microtubules are propelled faster with velocities equal to the maximal stepping velocity of individual motors. Because, in this case, the collective drag force on the motors even at very low slipping velocity, is large enough to balance the microtubule drag in the solution.
The theoretical model developed based on this explanation is in good agreement with the experimental data of gliding velocities at different motor densities. The model gives information about the distance that the diffusing motors can isotropically reach to bind to a microtubule, which for membrane-anchored kinesin-1 is ~0.3 µm, an order of magnitude higher as compared to rigidly bound motors, owing to the lateral mobility of motors on the membrane. In addition, the model can be used to predict the number of motors involved in transport of a microtubule based on its gliding velocity.
In the second part of the thesis, we investigated the effect of loose inter-motor coupling on the transport behavior of KIF16B, a recently discovered kinesin motor with an inherent lipid-binding domain. Recent studies based on cell biological and cell extract experiments, have postulated that cargo binding of KIF16B is required to activate and dimerize the motor, making it a superprocessive motor. Here, we demonstrate that recombinant full-length KIF16B is a dimer even in the absence of cargo or additional proteins. The KIF16B dimers are active and processive, which demonstrates that the motors are not auto-inhibited in our experiments. Thus, in cells and cell extracts Kif16B may be inhibited by additional factors, which are removed upon cargo binding. Single molecule analysis of KIF16B-GFP reveals that the motors are not superprocessive but exhibit a processivity similar to kinesin-1 indicating that additional factors are most likely necessary to achieve superprocessivity. Transport on membrane-anchored KIF16B motors exhibited a similar cooperative behavior as membrane-anchored kinesin-1 where the microtubule gliding velocity increased with increasing motor density.
Taken together, our results demonstrate that the loose coupling of motors via lipid bilayers provides flexibility to cytoskeletal transport systems and induces cooperativity in multi-motor transport. Moreover, our ‘membrane-anchored’ gliding motility assays can be used to study the effects of lipid diffusivity (e.g. the presence of lipid micro-domains and rafts), lipid composition, and adaptor proteins on the collective dynamics of different motors.:Abstract vii
1 Introduction 1
1.1 Intracellular transport driven by motor proteins 2
1.2 Attachment of motor proteins to cargo 13
1.3 In vitro approaches to study transport by motor proteins 16
1.4 Aim of this study 23
2 Transport by kinesin-1 anchored to supported lipid bilayers 24
2.1 Formation and characterization of biotinylated SLBs 26
2.2 Anchoring kinesin-1 to biotinylated SLBs 28
2.3 Gliding motility of microtubules by kinesin-1 linked to SLBs 34
2.4 Theoretical description of gliding motility on diffusing motor proteins 40
2.5 Comparison of the gliding velocity between experiment and theory 46
2.6 Gliding motility on phase-separated SLBs 53
2.7 Discussion 55
3 Transport by KIF16B with an inherent lipid-binding domain 62
3.2 Biophysical characterization of KIF16B 70
3.3 Gliding motility of microtubules by KIF16B linked to SLBs 78
3.4 Transport of SUVs and lipid-coated beads attached to KIF16B 87
3.5 Discussion 90
4 Conclusion and outlook 96
5 Materials and methods 99
5.1 Reagents and solutions 99
5.2 Molecular biology 100
5.3 Protein expression and purification 104
5.4 In vitro motility assays 110
5.5 Image acquisition and data analysis 118
References 126
List of figures 141
List of tables 143
Abbreviations and symbols 144
Acknowledgements 147
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa:de:qucosa:29376 |
| Date | 25 November 2015 |
| Creators | Grover, Rahul |
| Contributors | Diez, Stefan, Schwille, Petra, 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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