The force required to remove tubulin from the microtubule lattice by pulling on its α-tubulin C-terminal tail

Abstract

Severing enzymes and molecular motors extract tubulin from the walls of microtubules by exerting mechanical force on subunits buried in the lattice. However, how much force is needed to remove tubulin from microtubules is not known, nor is the pathway by which subunits are removed. Using a site-specific functionalization method, we applied forces to the C-terminus of α-tubulin with an optical tweezer and found that a force of ~30 pN is required to extract tubulin from the microtubule wall. Additionally, we discovered that partial unfolding is an intermediate step in tubulin removal. The unfolding and extraction forces are similar to those generated by AAA-unfoldases. Lastly, we show that three kinesin-1 motor proteins can also extract tubulin from the microtubule lattice. Our results provide the first experimental investigation of how tubulin responds to mechanical forces exerted on its α-tubulin C-terminal tail and have implications for the mechanisms of severing enzymes and microtubule stability.

The rate of microtubule breaking increases exponentially with curvature

“…Breaking while smoothly gliding could result from the gradual weakening of a microtubule due to the repeated forces exerted by kinesin motors on the microtubule 1 , 24 – 26 , the photodamage experienced while the microtubule is in the field-of-view 9 – 11 , and the strain on the microtubule lattice at locations of high curvature 13 , 15 .…”

Molecular motors enhance microtubule lattice plasticity

“…It has been suggested in Ref. [21], that the walk of single kinesins is sufficient to directly remove dimers from the MT shaft as a rare event. Indeed, the experiments in Ref.…”

“…Indeed, the experiments in Ref. [21] show that the cooperative action of several kinesins may be able to remove a tubulin dimer from the lattice by pulling on the dimer via a flexible tether, although a direct proof of dimer removal is missing. Within this concept of direct dimer removal, the motor walk facilitates vacancy nucleation, as an extremely rare event.…”

“…These point defects have been identified by scanning force microscopy of dynamic MTs [19] and it has already been speculated that long-lived point defects may serve as point of attack for MT severing enzymes [20]. A recent experiment suggests that the combined walk of several kinesins is able remove tubulin dimers from the lattice [21].…”

Running Kinesin-1 shapes the microtubule acetylation gradient

“…Damage sites along the microtubule can result from different phenomena: i) imperfect polymerization, ii) occur spontaneously, iii) mechanical forces, or iv) activity of proteins including severing enzymes. Molecular motors do not only use microtubules to transport their cargo, but also generate stress within the microtubule which can damage their underlying microtubule tracks [33][34][35][36][37][38][39] .…”