Cooperative stabilization of microtubule dynamics by EB1 and CLIP-170 involves displacement of stably bound P(i) at microtubule ends.

End binding protein 1 (EB1) and cytoplasmic linker protein of 170 kDa (CLIP-170) are two well-studied microtubule plus-end-tracking proteins (+TIPs) that target growing microtubule plus ends in the form of comet tails and regulate microtubule dynamics. However, the mechanism by which they regulate microtubule dynamics is not well understood. Using full-length EB1 and a minimal functional fragment of CLIP-170 (ClipCG12), we found that EB1 and CLIP-170 cooperatively regulate microtubule dynamic instability at concentrations below which neither protein is effective. By use of small-angle X-ray scattering and analytical ultracentrifugation, we found that ClipCG12 adopts a largely extended conformation with two noninteracting CAP-Gly domains and that it formed a complex in solution with EB1. Using a reconstituted steady-state mammalian microtubule system, we found that at a low concentration of 250 nM, neither EB1 nor ClipCG12 individually modulated plus-end dynamic instability. Higher concentrations (up to 2 μM) of the two proteins individually did modulate dynamic instability, perhaps by a combination of effects at the tips and along the microtubule lengths. However, when low concentrations (250 nM) of EB1 and ClipCG12 were present together, the mixture modulated dynamic instability considerably. Using a pulsing strategy with [γ(32)P]GTP, we further found that unlike EB1 or ClipCG12 alone, the EB1-ClipCG12 mixture partially depleted the microtubule ends of stably bound (32)P(i). Together, our results suggest that EB1 and ClipCG12 act cooperatively to regulate microtubule dynamics. They further indicate that stabilization of microtubule plus ends by the EB1-ClipCG12 mixture may involve modification of an aspect of the stabilizing cap.