Directed run-times are robust, but non-directed run-times are sensitive to cytoskeletal perturbation. (A) The directed run-time distribution indicates vesicles spend a similar amount of time undergoing directed motion independent of cytoskeletal perturbation. These durations are relatively short, typically less than 500 ms. (B) Non-directed run-times indicate two timescales of random motion, short (<3 s) and long (>25 s) for all conditions. When cytoskeletal perturbations are introduced, there is a shift in non-directed motion from short to long run-times. Inactivation of myosin-II has the strongest effect, shifting nearly half of vesicles to experience mainly non-directed motion.