Figure 4.

Importin-β regulates the microtubule-binding region that lies C-terminal to the NuMA NLS. GMPCPP-stabilized microtubules (X-rhodamine– and biotin-labeled) were incubated with NuMA-tail (1868–1997) and NuMA-tail (1970–2091) that contain microtubule-binding regions (light and dark gray) and NLS (black). (A and B) 20 nM and 800 nM of NuMA-tail (1868–1997) —GFP were examined under conditions of 1XBRB80 (A) and 0.25XBRB80 (B), respectively. (C and D) 20 nM of NuMA-tail (1970–2091)—GFP in the absence (C) or presence (D) of Importin-α/-β was analyzed. Bars, 10 µm. (E) Mean fluorescence signals under the different conditions shown in A–D were measured and plotted (n = 200 microtubules for each condition). SD was determined from data pooled from three independent experiments. Two-tailed Student t test; statistical differences: ***, P < 0.001. (F) The microtubule-binding region that lies C-terminal to the NLS (aa 1997–2101; brown dotted line) in NuMA is sterically blocked by Importin-β (light blue) that binds to the IBB domain of Importin-α (green; PDB code: 1QGK). Two microtubule-binding regions (MTBRs) are indicated.