Abstract
The motor proteins dynein and kinesin function collectively to achieve long-range, bidirectional transport along microtubules. Transport in live cells, imaged using phase microscopy, exhibits distinct modes of motility, with fast, unidirectional movement in both anterograde and retrograde directions as well as saltatory, bidirectional movement. To examine transport in a simplified environment, we isolated axonal transport vesicles from transgenic mice expressing GFP-dynamitin. The fluorescent vesicles were imaged with high resolution using total internal reflection fluorescence microscopy. Using automated tracking software, we tracked all vesicles associated with polarity-marked microtubules. The purified vesicles move bidirectionally with 40% of motility in the anterograde direction and 60% in the retrograde direction, similar to the bidirectional population of vesicles observed in live cells. Inhibitory antibodies to dynein modulate the direction of transport. We compared the predictions of a simple tug-of-war model, proposed by Muller et al., [PNAS, 2008] to the observed motility in vitro, and found good agreement when 6-7 dynein motors and 1 kinesin motor are active. This prediction is in striking agreement to quantitation of motor numbers through photobleaching and quantitative western blotting, which estimate approximately 6 dynein motors per vesicle and a ratio of 6.3±0.7 dynein motors to each kinesin-1 motor. Together, the analysis of vesicle transport in live cells, purified vesicles in vitro, and mathematical modeling indicate that vesicles move robustly with a small complement of motors. The results suggest an efficient regulatory scheme where small changes in the number of active motors manifest in large changes in the motility of the cargo.
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