ISWI assembles MTs in vitro, in a RanGTP-dependent manner. (A) ISWI bundles MTs in vitro. Recombinant ISWI (1.3 µM) was incubated with 0.3 µM taxol-stabilized MTs incorporating Cy3-labeled tubulin at RT for 20 min. (Top) The samples were squashed without fix solution and immediately imaged. Bar, 20 µm. Note that there are many single MTs in the absence of ISWI. (Bottom) The samples were stained with uranyl acetate and imaged by electron microscopy. Bar, 0.2 µm. (B) ISWI polymerizes MTs and forms asters in vitro. ISWI (2.0 µM) was incubated with 2.5 µM or 20 µM pure tubulin (10% Cy3-tubulin) at 37°C for 30 min. The samples were fixed, spun onto coverslips, and imaged. Bar, 20 µm. (C) EM analysis of the structures assembled in B with 2.5 µM tubulin and 2 µM ISWI. (Top) Low magnification image, showing MT asters. Bar, 2 µm. (Bottom) High magnification image of the dashed box in the top panel, showing plus-end MTs of the aster. Bar, 0.2 µm. (D) RanGTP activates ISWI to polymerize MTs. A CSF extract was incubated with rabbit IgG or anti–full-length ISWI-coated beads. The immunoprecipitates were incubated with 20 µM pure tubulin (2 µM Cy3-tubulin) in the presence or absence of RanQ69L at 37°C for 30 min. The samples were fixed, spun down onto coverslips, and imaged. Bar, 20 µm. (E) ISWI interacts with importin-α and -β. Control and ISWI immunoprecipitates were resolved by SDS-PAGE. (Top) Coomassie staining. (Bottom) Immunoblot with antibodies against ISWI, importin-β, or importin-α.