Figure 1.
NuMA is a dynein adaptor that requires dynactin and Lis1 to activate dynein motility. (A) Schematic of mScarlet-tagged NuMAFL and SNAP-tagged NuMAN-term constructs, indicating the main structural parts of NuMA (N-terminal region, predicted coiled-coil, C-terminal tail) and the domains or motifs implicated in dynein/dynactin-binding (Okumura et al., 2018; Renna et al., 2020). (B) Schematic of microscopy flow chambers (left), with functionalized glass surface and components of dynein motility assays (right). (C) Representative TIRF microscopy kymographs showing the motility of mEGFP-dynein in the presence of dynactin and AF546-NuMAN-term at different mCherry-Lis1 concentrations. Concentrations as indicated. (D) Processive run frequency of mEGFP-dynein (median ± 95% CI, estimated by bootstrapping). Each distinct color refers to a replicate; each data point represents the number of events in one field of view of one replicate; n = 99, 101, 27, 100, 29, 26, 103 microtubules. The grey curve represents a hyperbolic fit; apparent Kd and goodness-of-fit (expressed by R2) are indicated; dashed lines: 95% CI of the fit (estimated by bootstrapping). (E) Velocity distribution of processive mEGFP-dynein runs (mean of medians ± SEM). Each color refers to a replicate; each circle represents the median velocity of one replicate; n = 760, 3,751, 7,128, velocities; adjusted P values by Welch’s ANOVA with Holm-Sidak’s post-hoc test for multiple comparisons: 0.6121 (10 versus 100 nM), 0.6121 (10 versus 5,000 nM), and 0.6593 (100 versus 5,000 nM). Protein combination and concentrations in D and E as in C, and as indicated. (F) Representative kymographs showing the motility of mEGFP-dynein in the presence of dynactin and mCherry-Lis1 at different AF546-NuMAN-term concentrations. Concentrations as indicated. (G) Processive run frequency of mEGFP-dynein (median ± 95% CI). Symbols and curve as in D; n = 103, 70, 100, 70 microtubules. (H) Velocity distribution of processive mEGFP-dynein runs (mean of medians ± SEM). Circles as in E; n = 1,585, 3,482, 2,649 velocities; adjusted P values by Welch’s ANOVA with Holm-Sidak’s post-hoc test for multiple comparisons: 0.7632 (100 versus 200 nM), 0.7084 (10 versus 500 nM), and 0.9235 (200 versus 500 nM). Protein combinations and concentrations in G, H as in F, and as indicated. (I–K) Representative kymographs showing the motility of (I) mEGFP-dynein (left) and AF647-NuMAN-term (right) in the presence of dynactin and Lis1, (J) mEGFP-dynein in the presence of dynactin, Lis1, and mScarlet-NuMAFL, (K) mEGFP-dynein (left) and mScarlet-NuMAFL (right) in the presence of dynactin and Lis1. Arrowheads of the same color indicate co-localization in the same processive events. All data refer to motility on surface-immobilized Atto647N-labeled GMPCPP-microtubules (MTs) in dynein microscopy buffer. All data are from at least three biological replicates. Experiments shown in C‒H and J were carried out at 30°C, and those shown in I and K at 18°C.

NuMA is a dynein adaptor that requires dynactin and Lis1 to activate dynein motility. (A) Schematic of mScarlet-tagged NuMAFL and SNAP-tagged NuMAN-term constructs, indicating the main structural parts of NuMA (N-terminal region, predicted coiled-coil, C-terminal tail) and the domains or motifs implicated in dynein/dynactin-binding (Okumura et al., 2018; Renna et al., 2020). (B) Schematic of microscopy flow chambers (left), with functionalized glass surface and components of dynein motility assays (right). (C) Representative TIRF microscopy kymographs showing the motility of mEGFP-dynein in the presence of dynactin and AF546-NuMAN-term at different mCherry-Lis1 concentrations. Concentrations as indicated. (D) Processive run frequency of mEGFP-dynein (median ± 95% CI, estimated by bootstrapping). Each distinct color refers to a replicate; each data point represents the number of events in one field of view of one replicate; n = 99, 101, 27, 100, 29, 26, 103 microtubules. The grey curve represents a hyperbolic fit; apparent Kd and goodness-of-fit (expressed by R2) are indicated; dashed lines: 95% CI of the fit (estimated by bootstrapping). (E) Velocity distribution of processive mEGFP-dynein runs (mean of medians ± SEM). Each color refers to a replicate; each circle represents the median velocity of one replicate; n = 760, 3,751, 7,128, velocities; adjusted P values by Welch’s ANOVA with Holm-Sidak’s post-hoc test for multiple comparisons: 0.6121 (10 versus 100 nM), 0.6121 (10 versus 5,000 nM), and 0.6593 (100 versus 5,000 nM). Protein combination and concentrations in D and E as in C, and as indicated. (F) Representative kymographs showing the motility of mEGFP-dynein in the presence of dynactin and mCherry-Lis1 at different AF546-NuMAN-term concentrations. Concentrations as indicated. (G) Processive run frequency of mEGFP-dynein (median ± 95% CI). Symbols and curve as in D; n = 103, 70, 100, 70 microtubules. (H) Velocity distribution of processive mEGFP-dynein runs (mean of medians ± SEM). Circles as in E; n = 1,585, 3,482, 2,649 velocities; adjusted P values by Welch’s ANOVA with Holm-Sidak’s post-hoc test for multiple comparisons: 0.7632 (100 versus 200 nM), 0.7084 (10 versus 500 nM), and 0.9235 (200 versus 500 nM). Protein combinations and concentrations in G, H as in F, and as indicated. (I–K) Representative kymographs showing the motility of (I) mEGFP-dynein (left) and AF647-NuMAN-term (right) in the presence of dynactin and Lis1, (J) mEGFP-dynein in the presence of dynactin, Lis1, and mScarlet-NuMAFL, (K) mEGFP-dynein (left) and mScarlet-NuMAFL (right) in the presence of dynactin and Lis1. Arrowheads of the same color indicate co-localization in the same processive events. All data refer to motility on surface-immobilized Atto647N-labeled GMPCPP-microtubules (MTs) in dynein microscopy buffer. All data are from at least three biological replicates. Experiments shown in C‒H and J were carried out at 30°C, and those shown in I and K at 18°C.

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