GDP-tubulin assembles into microtubules that preferentially grow from the minus end and do not spontaneously depolymerize. (A) Diagram of the experimental procedure (see Materials and methods). S, seed. (B) Representative kymographs of microtubules grown from red seeds and green GTP-tubulin (6 µM) or GDP-tubulin (210 µM). (C) Two-step perfusion to identify the growing end of GDP-tubulin-assembled microtubules. GDP-tubulin (210 µM) is first perfused into the chamber, then GTP-tubulin (10 µM). (D) A representative kymograph of a growing microtubule recorded during the last step is shown in C (gray square). The green band corresponds to the microtubule that has grown during the first incubation with green GDP-tubulin. The growth rates of GTP-tubulin-assembled microtubules (magenta) identify microtubule polarity (see Materials and methods). (E) Comparison of minus-end dynamics of microtubules grown from GDP-tubulin (210 µM) or GTP-tubulin-grown (6 µM). Values represent mean ± SD. n, number of events from at least three independent experiments. (F) Growth rate of microtubule minus-end as a function of GDP-tubulin (red) and GTP-tubulin concentration (blue, inset). Values represent mean ± SD of at least 31 growth events for GDP-tubulin and 156 growth events for GTP-tubulin from at least three independent experiments. Triangles represent the mean growth rate for each experiment.