Abstract
The microtubule-associated protein, TPX2, regulates the activity of the mitotic kinesin, Eg5, but the mechanism of regulation is not established. Using total internal reflection fluorescence microscopy, we observed that Eg5, in extracts of mammalian cells expressing Eg5-EGFP, moved processively toward the microtubule plus-end at an average velocity of 14 nm/s. TPX2 bound to microtubules with an apparent dissociation constant of ∼ 200 nm, and microtubule binding was not dependent on the C-terminal tails of tubulin. Using single molecule assays, we found that full-length TPX2 dramatically reduced Eg5 velocity, whereas truncated TPX2, which lacks the domain that is required for the interaction with Eg5, was a less effective inhibitor at the same concentration. To determine the region(s) of Eg5 that is required for interaction with TPX2, we performed microtubule gliding assays. Dimeric, but not monomeric, Eg5 was differentially inhibited by full-length and truncated TPX2, demonstrating that dimerization or residues in the neck region are important for the interaction of TPX2 with Eg5. These results show that both microtubule binding and interaction with Eg5 contribute to motor inhibition by TPX2 and demonstrate the utility of mammalian cell extracts for biophysical assays.
Highlights
TPX2 is a mitotic microtubule-associated protein that regulates the kinesin, Eg5
We found that full-length TPX2 dramatically reduced Eg5 velocity, whereas truncated TPX2, which lacks the domain that is required for the interaction with Eg5, was a less effective inhibitor at the same concentration
TPX2 Binding to Microtubules—To examine the regulation of mammalian Eg5 by TPX2, we expressed and purified fulllength TPX2 and a truncated version lacking the C-terminal 35 amino acids that mediate the interaction with Eg5 (10, 11) (Fig. 1A)
Summary
TPX2 is a mitotic microtubule-associated protein that regulates the kinesin, Eg5. Results: Full-length TPX2 is a more potent inhibitor of Eg5 velocity than truncated TPX2; differential regulation by TPX2 was not observed for monomeric Eg5. But not monomeric, Eg5 was differentially inhibited by full-length and truncated TPX2, demonstrating that dimerization or residues in the neck region are important for the interaction of TPX2 with Eg5. These results show that both microtubule binding and interaction with Eg5 contribute to motor inhibition by TPX2 and demonstrate the utility of mammalian cell extracts for biophysical assays. Mitotic motor proteins play important roles in regulating microtubule organization and dynamics and in generating the forces required for spindle formation and chromosome motion. Our experiments provide new insight into the microtubule-associated protein TPX2 and its regulation of the mitotic kinesin Eg5
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