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Fig. 8. Xenopus NEDD1 (XNEDD1) bundles microtubules in vitro and localizes to kinetochore fibers in Xenopus tissue culture cells. (A) Recombinant XNEDD1-C (without the GST used to purify the protein) added to in vitro microtubule polymerization reactions causes microtubule bundling. Recombinant proteins (as indicated) were added to the reaction at the indicated concentrations. Scale bar, 10 µm. (B) Recombinant XNEDD1-C binds to microtubules along their length. (a) GST-XNEDD1-C was allowed to bind to microtubules, and the samples were processed for immunofluorescence with antibodies against GST (XNEDD1-C; green in overlay) or -tubulin (red in overlay). Scale bar, 5 µm. (b) Microtubule-pelleting assay using a constant amount of recombinant XNEDD-C and increasing amounts of taxol-stabilized microtubules. Supernatant (S) and pellet (P) of each sample were loaded on a gel and stained with Coomassie Blue. Only the part of the gel corresponding to XNEDD1 (upper panels) or tubulin (lower panels) are shown. (C) Recombinant XNEDD1-C bundles preformed microtubules. Recombinant XNEDD1-C (without the GST used to purify the protein) was incubated with taxol-stabilized microtubules. An aliquot of the reaction mixture was spotted onto a microscope slide and viewed under the microscope. Microtubules appear highly bundled in the presence of 300 nM XNEDD1-C. Scale bar, 10 µm. (D) XNEDD1 binds to kinetochore fibers. (Upper panels) Immunofluorescence shows that XNEDD1-positive fibers end at kinetochores stained by Bub1. XNEDD1, green; Bub1, red; DNA, blue. Scale bar, 5 µm. (Lower panels) Immuno-localization of XNEDD1 at kinetochore fibers. XNEDD1, green; kinetochore fibers, red; DNA, blue. Scale bar, 5 µm. (E) Higher magnification image of the boxed area shown in D.

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