Bidirectional transport of vesicles by dynein and kinesin

Intracellular transport is fundamental to many cellular processes- from capturing and destroying pathogens to the propagation of nerve impulses. This transport is mediated by specialized enzymes that convert the free energy of ATP hydrolysis to ‘walk’ on polymeric filaments. The microtubule filament and its associated motors- dynein and kinesin - are responsible for the long-range transport of various cellular cargoes. While these two motors move in the opposite directions, they are often simultaneously present on individual cargoes leading to bidirectional motility characterized by frequency directional reversals. How this process is regulated and what determines the direction of cargo transport remains poorly understood. Addressing these questions requires a systematic analysis where the contribution of various factors in regulating/determining the transport direction of a well-defined cargo can be elucidated. This project establishes an in vitro assay where we reconstitute bidirectional motility of large unilamellar vesicles driven by purified dynein and kinesin-3 motors. Vesicles exhibit fast runs in either direction, with a subset exhibiting directional reversals. The transport features of these vesicles are remarkably similar to that of cargoes in vivo and do not require any additional regulatory proteins/complexes. The simultaneous opposing activity of dynein and kinesin-3 leads to tugs-of-war for a finite period. Finally, we use this assay to determine how microtubule-associated proteins that have differential activity towards dynein and kinesin affect the transport direction of vesicles. MAP9 biases the direction of vesicles towards the plus-end by limiting the ability of dynein to land on microtubules. Our approach can be extended to investigate the potential biasing activities of cellular factors such as posttranslational modifications of tubulin, motor-adaptors, etc. and physical factors, such as fluidity and tension of the vesicle membrane.:1 Introduction 1.1 Microtubules, dynein, kinesin, and microtubule-associated proteins (MAPs) 1.2 Bidirectional transport: In vivo observations and models 1.3 Bidirectional transport: In vitro reconstitution 1.4 Role of membranes in modulating motor activities 1.5 Aims of the thesis 2 Purification of dynein and dynactin 2.1 Purification of dynein and dynactin from adherent mammalian cell cultures 2.2 Purification of dynein and dynactin from suspension cultures using BacMam system 2.3 Validation of dynein and dynactin function using single-molecule motility assays 3 Reconstitution and characterization of bidirectional vesicle motility 3.1 DDB-KIF16B-vesicles exhibit directional reversals in vitro 3.2 Transport direction of vesicles is dependent on the relative concentration of DDB and KIF16B 3.3 Opposing motors do not affect the velocity of the driving motor 3.4 DDB and KIF16B engage in a tug-of-war before directional reversals 3.5 Discussion 4 Characterization of bidirectional motility in the presence of MAPs 4.1 Tau does not differentially affect DDB versus KIF16B at the single-molecule level 4.2 MAP9 affects landing of DDB but not KIF16B 4.3 Motor ensembles can circumnavigate the inhibitory effects of MAPs 4.4 Tau does not bias the transport direction of DDB-KIF16B-vesicles 4.5 MAP9 biases the transport direction of DDB-KIF16B-vesicles towards the plus-end 4.6 Discussion 5 Conclusion and outlook 6 Materials and Methods 6.1 Molecular Biology 6.2 Culture of Flp-In 293 cells 6.3 Protein biochemistry 6.4 In vitro motility assays 6.5 Data processing and analysi