Phase Separation and a Hydrodynamic Instability Localize Proteins at Growing Microtubule Ends

Regulating microtubule dynamics is essential for cellular function, and precise localization of regulatory proteins at microtubule ends is critical. End-binding protein EB3, a key regulator of microtubule growth, accumulates over an extended region at the growing end, forming a comet that gradually fragments into transient droplets along the microtubule shaft. Here, we combine in vitro reconstitution experiments with theoretical analysis to show that surface-mediated condensation of EB3 effectively localizes the protein at microtubule ends. Our results reveal that a Rayleigh–Plateau instability limits the condensate’s extent, producing a finite comet length and discrete droplets along the shaft. Remarkably, the comet size is independent of the GTP–cap size. This finding is supported by experiments on cells showing that modulation of microtubule growth velocity and hence GTP–cap size do not consistently alter EB3 comet length. Furthermore, our theory shows that rapid droplet evaporation requires a transition of EB3 to a non–phase-separating state. Overall, our work challenges the view that comet length directly reflects GTP–cap size and highlights a novel mechanism for regulating microtubule dynamics.