Negative regulation of EB1 turnover at microtubule plus ends by interaction with microtubule-associated protein ATIP3.

Lauriane Velot,Benjamin Pierre Bouchet,Clara Nahmias,Laurence Serre,Ariel Savina,Stéphane Honore,Angie Molina,Sylvie Rodrigues-Ferreira,Marina Morel,Diane Braguer,Ludovic Leconte,Anne Nehlig,Isabelle Arnal

doi:10.18632/oncotarget.6196

Abstract

The regulation of microtubule dynamics is critical to ensure essential cell functions. End binding protein 1 (EB1) is a master regulator of microtubule dynamics that autonomously binds an extended GTP/GDP-Pi structure at growing microtubule ends and recruits regulatory proteins at this location. However, negative regulation of EB1 association with growing microtubule ends remains poorly understood. We show here that microtubule-associated tumor suppressor ATIP3 interacts with EB1 through direct binding of a non-canonical proline-rich motif. Results indicate that ATIP3 does not localize at growing microtubule ends and that in situ ATIP3-EB1 molecular complexes are mostly detected in the cytosol. We present evidence that a minimal EB1-interacting sequence of ATIP3 is both necessary and sufficient to prevent EB1 accumulation at growing microtubule ends in living cells and that EB1-interaction is involved in reducing cell polarity. By fluorescence recovery of EB1-GFP after photobleaching, we show that ATIP3 silencing accelerates EB1 turnover at microtubule ends with no modification of EB1 diffusion in the cytosol. We propose a novel mechanism by which ATIP3-EB1 interaction indirectly reduces the kinetics of EB1 exchange on its recognition site, thereby accounting for negative regulation of microtubule dynamic instability. Our findings provide a unique example of decreased EB1 turnover at growing microtubule ends by cytosolic interaction with a tumor suppressor.

Highlights

Microtubules (MTs) are polarized structures that continuously switch between periods of polymerization and depolymerization at their growing ends
We show that ATIP3 interacts with End binding protein 1 (EB1) in an MT-independent manner
To investigate whether ATIP3 interacts with EB1, we used anti-mCherry antibodies to isolate mChATIP3 complexes from MCF7 cells expressing mChATIP3 fusion protein and EB1 fused to green fluorescent protein (EB1-GFP)

Summary

Introduction

Microtubules (MTs) are polarized structures that continuously switch between periods of polymerization and depolymerization at their growing (plus) ends. Time-lapse analysis (Supplementary Figure S2A, Movies 1 and 2) clearly showed distinct patterns of mCh-ATIP3 and EB3-GFP localization in living cells and indicated that ATIP3 does not accumulate at growing MT ends.

Results

Conclusion