Competitive binding-mediated mesoscale protein-protein interactions direct microtubule growth

Abstract Microtubule organization in cells relies on targeting mechanisms. CLASP proteins are key regulators of microtubule organization, yet the underlying mechanisms remain elusive. Here, we revealed that the C-terminal domain of CLASP2 interacts with a common motif found in several CLASP2-binding proteins. This interaction drives the dynamic localization of CLASP2 to distinct cellular compartments, where CLASP2 accumulates in protein condensates at the cell cortex or the microtubule plus end. These condensates physically contact with each other via CLASP2-mediated competitive binding, determining cortical microtubule targeting. The phosphorylation of CLASP2 modulates the dynamics of the condensate-condensate interaction and spatiotemporally navigates microtubule growth. Moreover, we identified additional CLASP-interacting proteins that are involved in condensate contacts in a CLASP2-dependent manner, uncovering a general mechanism governing microtubule targeting. Our findings not only unveil a tunable multiphase system regulating microtubule organization, but also offers general mechanistic insights into intricate protein-protein interactions at the mesoscale level.