γTuRC-nucleated microtubules in the absence and presence of KIF2A. (A) Left: TIRF microscopy snapshot of single γTuRC-nucleated microtubules (magenta) 20 min after the start of imaging in the presence of 15 μM tubulin (AlexaFluor647, 5.4%). 1 nM biotinylated and BFP-tagged γTuRC (yellow) was used for immobilization. γTuRC signal is an average of all frames imaged during 20 min (right, top). The inset of the dashed area shows two γTuRC-capped microtubules. γTuRC-mediated nucleation sites are indicated by cyan arrows. Right, bottom: Kymograph showing microtubule (magenta) minus-end capping by γTuRC (yellow) and a dynamic plus end. (B) Plot of the percentage of spontaneously nucleated microtubules in solution in γTuRC nucleation assays using 12.5 μM of tubulin and different mScarlet-KIF2A (cyan) concentrations, as indicated. Number of microtubules analyzed per condition: n = 256; 20 nM, n = 249; 40 nM, n = 190; 100 nM, n = 91. Data for plots were pooled from three independent experiments. Error bars are SEM. For symbols without visible error bars, error bars are smaller than the symbol size. (C) Bar graph of the mean percentage of microtubules that either remain protected by γTuRC or are released after nucleation in the presence of the indicated concentrations of untagged KIF2A. Number of microtubules analyzed per condition: 0 nM, n = 162; 20 nM, n = 199; 100 nM, n = 83; Data for plots were pooled from at least two independent experiments. Error bars are SEM. 2 nM of γTuRC was used for immobilization in B and C.