EB 1 promotes microtubule dynamics and regulates tubulin sheet closure in vitro

1 EB1 promotes microtubule dynamics and regulates tubulin sheet closure in vitro. End binding 1 (EB1) is a plus-end-tracking protein (+TIP) that localizes to microtubule plus ends where it modulates their dynamics and interactions with intracellular organelles 1, 2. Although the regulating activity of EB1 on microtubule dynamics has been studied in cells 3-6 and purified systems 7-13 , the molecular mechanisms involved in its specific activity are still unclear. Here, we describe how EB1 regulates the dynamics and structure of microtubules assembled from pure tubulin. We found that EB1 stimulates microtubule spontaneous nucleation and growth rate, and promotes both catastrophes (transitions from growth to shrinkage) and rescues (reverse events). Electron cryomicroscopy showed that EB1 induces the initial formation of tubulin sheets that rapidly close into the common 13-protofilament-microtubule architecture. Our results suggest that EB1 favours the lateral association of free tubulin at microtubule sheet edges, thereby stimulating nucleation, sheet growth and closure. The reduction of sheet length at microtubule growing ends, together with the elimination of stressed microtubule lattices, may account for catastrophes. Conversely, occasional binding of EB1 to the microtubule lattice may induce rescues. Microtubules are unstable polymers that switch stochastically between growing and shrinking states, a particular behaviour known as dynamic instability 14. Essential for this process is the tubulin-bound GTP hydrolysis that occurs during microtubule assembly, since it creates an unstable lattice that can rapidly depolymerise 14. Microtubule growth involves the formation of outwardly curved tubulin sheets that gradually close into tubes, and which are thought to protect the lattice from catastrophe events 15, 16. Catastrophes may arise from loss of this capping structure through occasional closure of the sheets onto energetically unfavourable configurations 17. Conversely, rescues may involve a metastable intermediate state between disassembly and assembly 18. Catastrophes and rescues are finely regulated in cells and can be either stimulated or inhibited depending on the cell activity 14. Several proteins interact with unpolymerised tubulin and/or microtubules to modulate their dynamic properties 19. Amongst them, EB1 is a highly conserved +TIP that was first described as a partner of the tumour suppressor APC 1. EB1 accumulates at microtubule plus ends through a treadmilling process characterized by an association with the newly growing end coupled with a rapid dissociation from the lattice older part 2. The recognition of a specific structure at microtubule plus end (e.g. the growing tubulin sheet) may be the targeting mechanism involved, rather …