Mechanisms of microtubule dynamics from single-molecule measurements

Microtubules are dynamic polymers of αβ-tubulin heterodimers that organize the intracellular space and mediate faithful chromosome segregation. Microtubule function depends on dynamic instability, the apparently random GTPase-dependent switching between growing and shrinking. Microtubule dynamics derive from biochemical properties of individual tubulin subunits and how they interact with the polymer end, a complex environment where individual tubulins can have different numbers of neighbor contacts. A fundamental understanding of microtubule dynamics has been difficult to establish because of challenges measuring the number, strength, and nucleotide-dependence of tubulin binding sites on the microtubule end. We used an improved single-molecule assay to measure tubulin:microtubule interactions. In addition to the two expected classes of binding site (longitudinal and corner), we identified previously unrecognized third binding interaction. We further show that nucleotide state strongly influences the strength of inter-protofilament contacts, with little effect on intra-protofilament contacts, and that a mutation can modulate this nucleotide effect. By uncovering a new tubulin binding state on the microtubule end, clarifying how GDP influences microtubule stability, and demonstrating that the nucleotide effects are allosteric and tunable, these single-molecule measurements and accompanying computational simulations provide rich new biochemical insight into the fundamental mechanisms of microtubule dynamics.