Skip to main content
. Author manuscript; available in PMC: 2015 Apr 10.
Published in final edited form as: Cell. 2014 Apr 10;157(2):420–432. doi: 10.1016/j.cell.2014.02.018

Figure 1.

Figure 1

Characterizations of NuMA’s microtubule binding. (A) Schematic depicting full-length NuMA and the truncated NuMA-tail II-GFP used in this study. The microtubule binding domain is highlighted in green (aa: 1900–1970). (B–D) NuMA-tail II-GFP (2 nM, green) binds surface-attached microtubules (red), as visualized via TIRF microscopy (B, microtubules; C, NuMA-tail II-GFP; D, composite). (E–G) GFP alone (2 nM) shows no microtubule localization (Scale bars = 10 μm). (H) Western blot-based analysis (anti-GFP) of NuMA-tail II-GFP’s microtubule binding. NuMA-tail II-GFP concentration was 1 μM. (I) Band intensities from (H) were used to determine fraction of protein bound, and were plotted against microtubule concentration; data were fit to the Hill equation (binding affinity Kd = 1.2 ± 0.2 μM; Hill coefficient = 1.2 ± 0.1 (N = 3 independent experiments). (J) SDS-PAGE analysis of limited proteolysis reactions performed on NuMA-tail II-GFP with four unique proteases reveals multiple low-molecular weight digestion products. (K) The persistent bands at 26 kDa in the gels shown in (J) were confirmed to be GFP by Western blot. (L) Diffusion of NuMA-tail II-GFP on the microtubule lattice plotted as a kymograph (inset: microtubule, red; NuMA-tail II-GFP, green. Vertical scale bar = 5 s, Horizontal scale bar = 2 μm). Histogram of binding lifetimes is fit to a single exponential Ae-tτ(characteristic lifetime τ = 2.8 ± 0.2 s, N = 4 independent experiments, n = 1984 single particles). (M) The mean squared displacement of single molecule diffusion events is calculated for NuMA-tail II-GFP (n = 136 traces from N = 4 independent experiments). Fitting MSD = 2Dt + offset yields the diffusion constant, D = 53,700 ± 1,000 nm2/s. (N) Mean displacement analysis of the same diffusion events from (M) are fit to a linear relationship (slope = 0.8 ± 1.6 nm/s).