Figure 4.
NuMA caps and stabilizes dynamic microtubule minus ends. (A) Schematic of a two-flush TIRF microscopy assay: fluorescent tubulin is flowed into a channel containing surface-immobilized GMPCPP-seeds (first flush); microtubules are allowed to elongate from GMPCPP-seeds for ≈10 min; and tubulin is re-added together with fluorescent NuMA constructs (second flush). (B) Representative kymograph showing a control experiment with microtubule minus and plus ends (dim magenta) dynamically elongating from a surface-immobilized Atto647N-labeled GMPCPP-seed (bright magenta) in the presence of 10 µM Atto647N-tubulin; the dashed line marks the second flush of tubulin. (C, E, and G) Representative TIRF microscopy images (top) and kymographs (bottom) showing the growth behavior of microtubule ends (dim magenta) elongating from surface-immobilized Atto647N-labeled GMPCPP-seeds (bright magenta) in the presence of Atto647N-tubulin, before (above the dashed line) or after (below the dashed line) the addition of different mScarlet-tagged NuMA constructs (green) at different concentrations. Related to Video 1. (D, F, and H) Growth velocity distributions of minus and plus ends elongating from GMPCPP-seeds in the presence of 10 µM Atto647N-tubulin before (circles) and after (triangles) the addition of different mScarlet-tagged NuMA constructs at different concentrations (mean of medians ± SEM). Each big symbol represents the median velocity of one replicate and each small symbol represents the growth velocity of one microtubule end segment; D: n = 72, 73, 67; P values: 0.0572, 0.0111, 0.0012 (left plot) and 0.1221, 0.4972, 0.0008 (right plot). F: n = 72, 51, 66 (left plot) and 72, 53, 66 (right plot); P values: 0.0572, 0.0196, 0.0027 (left plot) and 0.1221, 0.9029, 0.4875 (right plot). H: n = 72, 57, 72 (left and right plots); P values: 0.0572, 0.0038, 0.0175 (left plot) and 0.1221, 0.6881, 0.1384 (right plot). All data are from three biological replicates; P values were calculated by paired t test comparing the “pre-NuMA addition” and “post-NuMA addition” for each condition. All experiments were performed in NuMA microscopy buffer.

NuMA caps and stabilizes dynamic microtubule minus ends. (A) Schematic of a two-flush TIRF microscopy assay: fluorescent tubulin is flowed into a channel containing surface-immobilized GMPCPP-seeds (first flush); microtubules are allowed to elongate from GMPCPP-seeds for ≈10 min; and tubulin is re-added together with fluorescent NuMA constructs (second flush). (B) Representative kymograph showing a control experiment with microtubule minus and plus ends (dim magenta) dynamically elongating from a surface-immobilized Atto647N-labeled GMPCPP-seed (bright magenta) in the presence of 10 µM Atto647N-tubulin; the dashed line marks the second flush of tubulin. (C, E, and G) Representative TIRF microscopy images (top) and kymographs (bottom) showing the growth behavior of microtubule ends (dim magenta) elongating from surface-immobilized Atto647N-labeled GMPCPP-seeds (bright magenta) in the presence of Atto647N-tubulin, before (above the dashed line) or after (below the dashed line) the addition of different mScarlet-tagged NuMA constructs (green) at different concentrations. Related to Video 1. (D, F, and H) Growth velocity distributions of minus and plus ends elongating from GMPCPP-seeds in the presence of 10 µM Atto647N-tubulin before (circles) and after (triangles) the addition of different mScarlet-tagged NuMA constructs at different concentrations (mean of medians ± SEM). Each big symbol represents the median velocity of one replicate and each small symbol represents the growth velocity of one microtubule end segment; D: n = 72, 73, 67; P values: 0.0572, 0.0111, 0.0012 (left plot) and 0.1221, 0.4972, 0.0008 (right plot). F: n = 72, 51, 66 (left plot) and 72, 53, 66 (right plot); P values: 0.0572, 0.0196, 0.0027 (left plot) and 0.1221, 0.9029, 0.4875 (right plot). H: n = 72, 57, 72 (left and right plots); P values: 0.0572, 0.0038, 0.0175 (left plot) and 0.1221, 0.6881, 0.1384 (right plot). All data are from three biological replicates; P values were calculated by paired t test comparing the “pre-NuMA addition” and “post-NuMA addition” for each condition. All experiments were performed in NuMA microscopy buffer.

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