Figure 5.
γTuRC prevents NuMA accumulation at microtubule minus ends. (A) Schematic of a γTuRC nucleation assay performed in the presence of mScarlet-NuMAFL. (B) Representative TIRF microscopy kymographs showing microtubules nucleated by surface-immobilized mBFP-γTuRC, and spontaneously nucleated microtubules in solution in the presence of 10 µM Atto647-tubulin and 5 nM mScarlet-NuMAFL. γTuRC and solution-nucleated microtubules can be distinguished because the latter diffuse on the surface. γTuRC-nucleated microtubules only occasionally display weak NuMA fluorescence at their minus ends (second kymograph), whereas the minus ends of solution-nucleated microtubules frequently show intense NuMA signals (third and fourth kymographs). (C) Frequency of microtubule minus end localization of NuMA on γTuRC-nucleated and spontaneously nucleated microtubules in the presence of 10 µM Atto647-tubulin and 5, 15, and 30 nM mScarlet-NuMAFL (mean ± SEM); each diamond represents the mean frequency of each condition, each distinct small symbol represents the frequency of a single replicate within a condition; bottom n = 80, 101, 104 microtubules; top n = 21, 54, 52 microtubules. For γTuRC-nucleated microtubules, cases in which NuMA localization at the minus end coincided with NuMA/γTuRC co-localization prior to nucleation were excluded from the analysis. (D) Background-corrected maximum intensity of mScarlet-NuMAFL at 30 nM localizing to either γTuRC-nucleated microtubule minus ends, spontaneously nucleated microtubule minus ends, or at randomly chosen microtubule-free surface areas (mean of medians ± SEM), in the presence of 10 µM Atto647-tubulin. Each big symbol represents the median of one replicate; each small symbol represents the intensity at one microtubule minus end or surface area; each replicate is represented by a distinct symbol shape (circle, diamond, triangle, square); n = 68, 18, 12 intensities; adjusted P values by Welch’s ANOVA test with Holm-Sidak’s post-hoc test for multiple comparisons: 0.0295 (solution versus γTuRC), 0.0295 (solution versus surface), 0.3793 (γTuRC versus surface). (E and F) Plots showing an increase of the solution-nucleated (E) and γTuRC-nucleated (F) microtubule number over time, in the presence of 0–30 nM mScarlet-NuMAFL (mean ± SEM). NuMA promotes spontaneous nucleation of microtubules in solution in a dose-dependent manner (E), without exerting any effect on γTuRC-mediated nucleation (F). Experiments were performed in γTuRC microscopy buffer. All data are from at least three biological replicates.

γTuRC prevents NuMA accumulation at microtubule minus ends. (A) Schematic of a γTuRC nucleation assay performed in the presence of mScarlet-NuMAFL. (B) Representative TIRF microscopy kymographs showing microtubules nucleated by surface-immobilized mBFP-γTuRC, and spontaneously nucleated microtubules in solution in the presence of 10 µM Atto647-tubulin and 5 nM mScarlet-NuMAFL. γTuRC and solution-nucleated microtubules can be distinguished because the latter diffuse on the surface. γTuRC-nucleated microtubules only occasionally display weak NuMA fluorescence at their minus ends (second kymograph), whereas the minus ends of solution-nucleated microtubules frequently show intense NuMA signals (third and fourth kymographs). (C) Frequency of microtubule minus end localization of NuMA on γTuRC-nucleated and spontaneously nucleated microtubules in the presence of 10 µM Atto647-tubulin and 5, 15, and 30 nM mScarlet-NuMAFL (mean ± SEM); each diamond represents the mean frequency of each condition, each distinct small symbol represents the frequency of a single replicate within a condition; bottom n = 80, 101, 104 microtubules; top n = 21, 54, 52 microtubules. For γTuRC-nucleated microtubules, cases in which NuMA localization at the minus end coincided with NuMA/γTuRC co-localization prior to nucleation were excluded from the analysis. (D) Background-corrected maximum intensity of mScarlet-NuMAFL at 30 nM localizing to either γTuRC-nucleated microtubule minus ends, spontaneously nucleated microtubule minus ends, or at randomly chosen microtubule-free surface areas (mean of medians ± SEM), in the presence of 10 µM Atto647-tubulin. Each big symbol represents the median of one replicate; each small symbol represents the intensity at one microtubule minus end or surface area; each replicate is represented by a distinct symbol shape (circle, diamond, triangle, square); n = 68, 18, 12 intensities; adjusted P values by Welch’s ANOVA test with Holm-Sidak’s post-hoc test for multiple comparisons: 0.0295 (solution versus γTuRC), 0.0295 (solution versus surface), 0.3793 (γTuRC versus surface). (E and F) Plots showing an increase of the solution-nucleated (E) and γTuRC-nucleated (F) microtubule number over time, in the presence of 0–30 nM mScarlet-NuMAFL (mean ± SEM). NuMA promotes spontaneous nucleation of microtubules in solution in a dose-dependent manner (E), without exerting any effect on γTuRC-mediated nucleation (F). Experiments were performed in γTuRC microscopy buffer. All data are from at least three biological replicates.

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