PLUS-END MOTORS OVERRIDE MINUS-END MOTORS DURING TRANSPORT OF SQUID AXON VESICLES ON MICROTUBULES

Plus- and minus-end vesicle populations from squid axoplasm were isola ted from each other by selective extraction of the minus-end vesicle m otor followed by 5'-adenylyl imidodiphosphate (AMP-PNP)induced microtu bule affinity purification of the plus-end vesicles. In the presence o f cytosol containing both plus- and minus-end motors, the isolated pop ulations moved strictly in opposite directions along microtubules in v itro. Remarkably, when treated with trypsin before incubation with cyt osol, purified plus-end vesicles moved exclusively to microtubule minu s ends instead of moving in the normal plus-end direction. This revers al in the direction of movement of trypsinized plus-end vesicles, in l ight of further observation that cytosol promotes primarily minus-end movement of liposomes, suggests that the machinery for cytoplasmic dyn ein-driven, minus-end vesicle movement can establish a functional inte raction with the lipid bilayers of both vesicle populations. The addit ional finding that kinesin overrides cytoplasmic dynein when both are bound to bead surfaces indicates that the direction of vesicle movemen t could be regulated simply by the presence or absence of a tightly bo und, plus-end kinesin motor; being processive and tightly bound, the k inesin motor would override the activity of cytoplasmic dynein because the latter is weakly bound to vesicles and less processive. In suppor t of this model, it was found that (a) only plus-end vesicles copurifi ed with tightly bound kinesin motors; and (b) both plus- and minus-end vesicles bound cytoplasmic dynein from cytosol.