Molecular motors orchestrate pause-and-run dynamics to facilitate intracellular transport

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, either by severing the motor-cargo link or by chemically modifying the microtubule track, vesicles move slower and become trapped in longer, less mobile pauses, failing to reach their destination. Our work redefines transport pauses as an essential, motor-driven part of the delivery process, revealing a fundamental principle that ensures robust cargo transport through the crowded cellular environment.