Abstract
Intracellular transport is essential for cellular organization and function. This process is driven by molecular motors that ferry cargo along microtubules, but is characterized by intermittent motility, where cargoes switch between directed runs and prolonged pauses. The fundamental nature of these pauses has remained a mystery, specifically whether they are periods of motor detachment and passive drifting or states of active motor engagement. By combining single-particle tracking with large-scale motion analysis, we discovered that pauses are not passive. Instead, they are active, motor-driven states. We uncovered a unifying quantitative law: the diffusivity of a vesicle during a pause scales with the square of its velocity during a run. This parabolic relationship, D(eff) ∝ v(2), holds true for both kinesin and dynein motors, different cargo types, and a variety of cellular perturbations. We show that this coupling arises because the number of engaged motors governs motility in both states. When we reduce motor engagement, vesicles move more slowly and become trapped in longer, less mobile pauses, collectively causing them to fail to reach their destination. Our work redefines transport pauses as an essential, motor-driven part of microtubule-based cargo delivery, revealing a quantitative principle that contributes to robust cargo transport through the crowded cellular environment.