The force required to remove tubulin from the microtubule lattice

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 unknown, as is the pathway by which subunits are removed. Using a site-specific functionalization method, we applied forces to the C-terminus of -tubulin with an optical tweezer and found that a force of [~]30 pN is required to extract tubulin from the microtubule wall. Consistent with this force, we show that several kinesins can also extract tubulin. 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, suggesting that severing proteins such as spastin and katanin use an unfoldase mechanism. Our results reveal the response of tubulin to mechanical force and advance our understanding of severing enzymes and microtubule stability.