EB proteins track the ends of growing microtubules and regulate microtubule dynamics both directly and by acting as the hub of the tip-tracking network. Mammalian cells express cell type-specific combinations of three EB proteins with different cellular roles. Here, we reconstitute EB1, EB2 and EB3 tip tracking in vitro . We find that all three EBs show rapid exchange at the microtubule tip and that their signal correlates to the microtubule assembly rate. However, the three signals differ in their maxima and position from the microtubule tip. Using microtubules built with nucleotide analogues and site-directed mutagenesis, we show that EB2 prefers binding to microtubule lattices containing a 1:1 mixture of different nucleotides and its distinct binding specificity is conferred by amino acid substitutions at the right-hand-side interface of the EB microtubule-binding domain with tubulin. Our data are consistent with the model that all three EB paralogues sense the nucleotide state of both β-tubulins flanking their binding site. Their different profile of preferred binding sites contributes to occupying spatially distinct domains at the temporally evolving microtubule tip structure.

Summary Microtubule (MT) plus-end tracking proteins (+TIPs) preferentially localize to MT plus-ends. End-binding proteins (EBs) are master regulators of the +TIP complex; however, it is unknown whether EBs are regulated by other +TIPs. Here, we show that Cytoplasmic linker associated proteins (CLASPs) modulate EB localization at MTs. In CLASP-depleted cells, EBs localized along the MT lattice in addition to plus-ends. The MT-binding region of CLASP was sufficient for restoring normal EB localization, while neither EB-CLASP interactions nor EB tail-binding proteins are involved. In vitro assays revealed that CLASP directly functions to remove EB from MTs. Importantly, this effect occurs specifically during MT polymerization, but not at pre-formed MTs. Increased GTP-tubulin content within MTs in CLASP-depleted cells suggests that CLASPs facilitate GTP-hydrolysis to reduce EB lattice binding. Together, these findings suggest that CLASPs influence the MT lattice itself to regulate EB and determine exclusive plus-end localization of EBs in cells.