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First published online November 3, 2003
doi: 10.1242/10.1242/jcs.00798

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Ran modulates spindle assembly by regulating a subset of TPX2 and Kid activities including Aurora A activation

Department of Medical Genetics and Microbiology, University of Toronto, 1 King's College Circle, Toronto, Ontario, Canada, M5S 1A8

View larger version (39K): [in a new window]   Fig. 1. The amino-terminal domain of TPX2 contains a nuclear localization signal and a microtubule-binding site. Fragments of TPX2 were fused to EGFP, expressed in HeLa cells and then fixed 24 hours after transfection. Microtubules were visualized using an anti-tubulin antibody (YOL1/34), DNA was visualized with DAPI, and the whole cells were viewed by differential interference contrast (DIC) microscopy. (A) TPX2 fragments fused to EGFP that localize to the nucleus (TPX2, TNT and 1T) or the cytoplasm (3T and 5T). (B) EGFP-TPX2 fragments that co-localize with microtubules (MT). (C) Summary outlining the sub-cellular localizations of the different fragments of TPX2 (amino acid residues in parenthesis), N, nuclear localization; C, cytosolic localization; box depicts the NLS-containing domain. Scale bar: 10 µm.

View larger version (28K): [in a new window]   Fig. 2. TPX2 can bind to the importin /ß complex, an interaction that prevents TPX2 binding to Aurora A, but not microtubules. (A) The amino terminal domain of TPX2, TNT or BSA was coupled to Affiprep 10 beads, incubated with mitotic HeLa cell extract in the presence or absence of the importin /ß complex and GST-RanL43E, then re-isolated and analyzed by SDS PAGE and immunoblotting to detect importin ß. (B) GST-TNT was incubated with Taxol-stabilized microtubules in the presence or absence of the recombinant GST-RanL43E and or the importin /ß complex. Microtubules were re-isolated by centrifugation and the subsequent pellet (P) and supernatant (S) fractions analyzed by SDS-PAGE and immuoblotting to detect GSTTNT. (C) S-tagged TNT was incubated with mitotic HeLa cell extract in the presence of absence of GST-RanL43E and the importin /ß complex. TNT was recovered from the extract with S-protein agarose beads and analyzed by SDS PAGE and immunoblotting to detect Aurora A.

View larger version (45K): [in a new window]   Fig. 3. The importin /ß complex prevents the recruitment of Aurora A to microtubules and its activation. (A) Taxol stabilized microtubules (MT) were incubated with mitotic HeLa cell extract in the presence or absence of GST-RanL43E and the importin /ß complex then re-isolated by centrifugation and the pellet fractions analyzed by SDS-PAGE and immunoblotting to detect TPX2 and Aurora A. (B) Taxol stabilized microtubules were incubated with mitotic HeLa cell extract in the presence or absence of GST-RanL43E or the importin /ß complex. After 30 minutes the extracts were analyzed by SDS-PAGE and immunoblotting to detect Aurora A phosphorylated on amino acid residue threonine 288 (P-Aurora A), then stripped and re-probed to detect all classes of Aurora A.

View larger version (50K): [in a new window]   Fig. 4. Kid has 3 NLS-containing domains. Fragments of Kid were fused to EGFP and expressed in HeLa cells and then fixed 24 hours after transfection. DNA was visualized with DAPI and the cells viewed by differential interference contrast (DIC) microscopy. The sub-cellular localization of the different Kid fragments is summarized in the table. N, nuclear; C, cytosolic; open box, kinesin homology domain; hatched box, DNA binding domain. Scale bar: 10 µm.

View larger version (37K): [in a new window]   Fig. 5. The microtubule-binding activity, but not the DNA binding activity of Kid is inhibited by the importin /ß complex. (A) The amino-terminal domain (KNT) and the carboxy-terminal domain (KCT) of Kid and BSA were immobilized onto Affiprep 10 beads, incubated with mitotic HeLa cell extract then re-isolated and analyzed by SDS-PAGE and immunoblotting to detect importin ß. (B) GST-KCT was incubated with DNA cellulose at 4°C for 1 hour and then successively washed with buffers containing 50 mM NaCl (50), 300 mM NaCl (300) and 500 mM NaCl (500). The eluates were analyzed by SDS-PAGE and Coomassie Blue staining. L, load. (C) GST-KCT was incubated with DNA cellulose in the presence or absence of GST-RanL43E and the importin /ß complex. The DNA cellulose was washed with 50 mM NaCl followed by 500 mM NaCl. The initial amount of KCT (L) incubated with the DNA cellulose was compared to that eluted with 500 mM NaCl (E). (D) GST-KNT was incubated with Taxol-stabilized microtubules in the presence or absence of GST-RanL43E and the importin /ß complex. The microtubules were re-isolated by centrifugation and the subsequent pellet (P) and supernatant (S) fractions analyzed by SDS-PAGE and immunoblotting to detect GST.