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Cytoplasmic dynein 1 is almost exclusively responsible for intracellular transport toward the minus-end of microtubules in animal cells. One of the key factors for the unidirectional movement of dynein is the asymmetry of the unbinding of the motor from the microtubule when an external load is applied; it dissociates more easily from microtubules with minus-end directed loading than with plus-end directed loading. To elucidate the molecular basis for this property, we performed molecular dynamics simulations to identify the key residues responsible for asymmetry, which were then examined experimentally. First, we reproduced asymmetry in the unbinding behavior of dynein using coarse-grained simulations. Then, data analysis together with mutational analysis in silico predicted the specific residues that may be responsible for the asymmetry in unbinding. To examine this prediction, we expressed and purified recombinant dynein with mutations in either of the identified key residues. Consistent with the simulations, one of the mutants did not exhibit asymmetry in the in vitro unbinding assay. Moreover, the mutant dynein was able to bind and move diffusely along a microtubule but was unable to restrict its movement to the minus-end direction. Our results demonstrate both experimentally and theoretically how the key residue on the microtubule-binding domain generates asymmetry in unbinding, which is a critical mechanism for the unidirectional movement of dynein along a microtubule track.Significance StatementCytoplasmic dynein moves to the minus end of microtubules. This unidirectional dynein motility provides the driving force for various cellular activities including vesicle transport, organelle positioning and cell division. One of the key factors for dynein to exhibit unidirectional movement is the asymmetry of unbinding of dynein from the microtubule depending on the direction of external load. By combining computational simulations and in vitro experiments, we identified a residue responsible for the asymmetry. A point mutation at the residue indeed abolished unidirectional motility, highlighting the importance of the asymmetric unbinding property in dynein’s unidirectional movement. |
