Distinct liquid-liquid phase separation properties of end-binding proteins EB1 and EB3

End-binding proteins (EBs) are central components of the network of microtubule-plus-end-tracking proteins (+TIPs) that modulate microtubule dynamics. Recent studies have shown that EBs undergo liquid-liquid phase separation (LLPS), and it was proposed that the resulting condensates could play a major role in the recruitment of other + TIPs as well as in polymerization of tubulin. Here, we performed detailed studies of LLPS properties of two major members of the EB family in mammalian cells, EB1 and EB3. Surprisingly, we found that, despite 67% sequence identity, EB3 has a significantly higher LLPS propensity than EB1, both in vitro and in cells. This difference is due to combined contributions from multiple protein regions, with histidine residues in the N-terminal domain playing a particularly important role. Furthermore, EB1 and EB3 condensates were found to differ in their material properties, with EB3 droplets being much less dynamic than the EB1 counterparts. Importantly, EB3 droplets had higher capacity to recruit tubulin and nucleate its polymerization. The differences with regard to the impact of condensation on tubulin polymerization were especially striking in the presence of another + TIP-associated protein, CLIP-170, in which case higher tubulin polymerization capacity was observed for EB3/CLIP-170 droplets than the EB1/CLIP-170 counterparts, and this difference was attributed to distinct material properties of the two droplet types. These findings suggest that different, EB-dependent + TIP body types may exist in cells, contributing to functional specialization of microtubules.