Figure 5.
γTuRC-nucleated microtubules are uncapped by KIF2A and spastin allowing microtubule treadmilling. (A) Time sequence of TIRF microscopy images and kymographs of microtubules (magenta) nucleated by surface-immobilized γTuRC in the presence of 11 µM Cy5-tubulin (5.4% labeling percentage), which remain unaffected by 100 nM of spastin. Arrowheads point to γTuRC-capped microtubule minus ends. (B) Time sequence of TIRF microscopy images and kymograph of γTuRC-nucleated microtubules (magenta) that were severed in the presence of 11 µM Cy5-tubulin, 20 nM mScarlet-KIF2A (cyan), and 100 nM of spastin. Yellow arrowheads and asterisks indicate γTuRC-capped microtubule minus-ends and severing events, respectively. (C) Time sequence of TIRF microscopy images and kymographs of γTuRC-nucleated microtubules (magenta) that were released from γTuRC by the combined action of mScarlet-KIF2A (cyan) and spastin. Conditions as in B. Yellow arrowheads point to the γTuRC-nucleation sites of microtubules and yellow asterisks indicate the release of the microtubules from γTuRC, leaving the minus end uncapped for depolymerization by mScarlet-KIF2A (cyan). (D) Percentage of microtubules that either remain protected by γTuRC or are released after nucleation for the indicated concentrations of mScarlet-KIF2A and spastin. Number of microtubules analyzed per condition: mScarlet-KIF2A and spastin: 0 nM, n = 228; mScarlet-KIF2A 0 nM and spastin 100 nM, n = 152; mScarlet-KIF2A 20 nM and spastin 0 nM, n = 160; mScarlet-KIF2A 20 nM and spastin 100 nM, n = 222. Data for plots were pooled from at least two independent experiments. Error bars are SEM. (E) Percentage of released and severed γTuRC-nucleated microtubules for the indicated concentration of spastin and mScarlet-KIF2A. Number of microtubules analyzed per condition: mScarlet-KIF2A 20 nM and spastin 100 nM, n = 222. Error bars are SEM. In all experiments, 2.5 nM γTuRC was used for surface immobilization. The time stamps indicate min:s.

γTuRC-nucleated microtubules are uncapped by KIF2A and spastin allowing microtubule treadmilling. (A) Time sequence of TIRF microscopy images and kymographs of microtubules (magenta) nucleated by surface-immobilized γTuRC in the presence of 11 µM Cy5-tubulin (5.4% labeling percentage), which remain unaffected by 100 nM of spastin. Arrowheads point to γTuRC-capped microtubule minus ends. (B) Time sequence of TIRF microscopy images and kymograph of γTuRC-nucleated microtubules (magenta) that were severed in the presence of 11 µM Cy5-tubulin, 20 nM mScarlet-KIF2A (cyan), and 100 nM of spastin. Yellow arrowheads and asterisks indicate γTuRC-capped microtubule minus-ends and severing events, respectively. (C) Time sequence of TIRF microscopy images and kymographs of γTuRC-nucleated microtubules (magenta) that were released from γTuRC by the combined action of mScarlet-KIF2A (cyan) and spastin. Conditions as in B. Yellow arrowheads point to the γTuRC-nucleation sites of microtubules and yellow asterisks indicate the release of the microtubules from γTuRC, leaving the minus end uncapped for depolymerization by mScarlet-KIF2A (cyan). (D) Percentage of microtubules that either remain protected by γTuRC or are released after nucleation for the indicated concentrations of mScarlet-KIF2A and spastin. Number of microtubules analyzed per condition: mScarlet-KIF2A and spastin: 0 nM, n = 228; mScarlet-KIF2A 0 nM and spastin 100 nM, n = 152; mScarlet-KIF2A 20 nM and spastin 0 nM, n = 160; mScarlet-KIF2A 20 nM and spastin 100 nM, n = 222. Data for plots were pooled from at least two independent experiments. Error bars are SEM. (E) Percentage of released and severed γTuRC-nucleated microtubules for the indicated concentration of spastin and mScarlet-KIF2A. Number of microtubules analyzed per condition: mScarlet-KIF2A 20 nM and spastin 100 nM, n = 222. Error bars are SEM. In all experiments, 2.5 nM γTuRC was used for surface immobilization. The time stamps indicate min:s.

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