NuMA gradually accumulates at the minus ends of enzymatically γTuRC-uncapped microtubule s . (A) Schematic of a γTuRC nucleation assay performed in the presence of KIF2A, spastin, and mScarlet-NuMA^FL. The combined action of spastin and KIF2A triggers γTuRC release, which is followed by microtubule treadmilling and minus end capping by NuMA. (B) Representative TIRF microscopy kymographs showing microtubules nucleated by surface-immobilized mBFP-γTuRC in the presence of 11 µM Atto647-tubulin, 20 nM KIF2A, 10 nM spastin, and 30 nM mScarlet-NuMA^FL. γTuRC-capped microtubule minus ends are stabilized, while released microtubule minus ends typically depolymerize under the action of KIF2A, which also increases the catastrophe frequency at the plus end, often resulting in early microtubule disappearance. After some time, NuMA can cap the minus ends of released microtubules, arresting treadmilling and increasing their lifetimes. (C) Representative TIRF microscopy time course images of microtubule minus ends stabilized by γTuRC (yellow circles) undergoing γTuRC release, consequential treadmilling (white dashed circles), and eventually NuMA capping (green circles). Protein concentrations as in B; timestamps refer to mm:ss; related to Video 2. (D) Frequency of different γTuRC-nucleated microtubule populations (mean ± SEM). Each distinct symbol represents a replicate; protein concentrations as in B; n = 349 γTuRC-nucleated microtubules. (E) Lifetime of γTuRC-nucleated microtubules following their release, in the absence or presence of NuMA capping their minus ends (means of medians ± SEM). Each big circle represents the median value of one replicate, each small symbol represents the lifetime of one microtubule of one replicate; n = 133 γTuRC released microtubules; P value by Welch’s t test: 0.0030. Experiments were performed in γTuRC release buffer. All data are from three biological replicates.