Single-molecule microtubule dynamics measurements reveal an intermediate state and clarify the role of nucleotide

Microtubules are polymers of αβ-tubulin heterodimers that undergo GTPase dependent polymerization dynamics to organize the intracellular space and mediate faithful chromosome segregation. 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 strength, nucleotide-dependence, and proportion of different tubulin binding sites on the microtubule end. Using an improved single-molecule assay to measure tubulin:microtubule interactions, we discover that tubulin can isomerize to bind more tightly to the microtubule end. By inferring binding affinities from tubulin off-rates, we find that nucleotide state strongly influences the strength of lateral contacts between protofilaments, with little effect on longitudinal contacts along protofilaments. A hyperstabilizing mutation modulates this nucleotide effect. By uncovering an additional 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 biochemical insight into the fundamental mechanisms of microtubule dynamics.