TPX2 Inhibits Eg5 by Interactions with Both Motor and Microtubule

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.

Keywords: Eg5; TPX2; kinesin; microtubule; microtubule-associated protein (MAP); mitosis; mitotic spindle; spindle.

FIGURE 1.

Binding of TPX2 and TPX2-710 to microtubules. A, schematic diagram of the TPX2 constructs (left) and Coomassie Brilliant Blue-stained gel of the purified proteins (right). B, co-sedimentation of TPX2 with microtubules. S, supernatant; P, pellet. The concentration of microtubules in each pair of lanes is noted above. Western blots were stained for TPX2 or tubulin. C, quantification of apparent affinity was performed using a quadratic fit. The experiment was performed twice, and the values were averaged. Error bars, S.D.

FIGURE 2.

Binding Dynamics of TPX2 and TPX2-710. A, box plot showing release of TPX2 and TPX2–710 from microtubules in the presence of the indicated concentration of KCl added to the buffer. TPX2 fluorescence is reported as arbitrary units (A.U.). Whiskers define the range, boxes encompass the 25th to 75th quartiles, and lines depict the medians. B, TPX2 and TPX2-710 binding to untreated and subtilisin A-digested microtubules; top panels, fluorescence images of TPX2-Halo or TPX2–710-Halo bound to untreated and subtilisin A-digested microtubules; middle, quantification of TPX2 fluorescence; bottom, polyacrylamide gel showing digested and control microtubules. TPX2 fluorescence was measured for at least 60 microtubules for each of two independent experiments; error bars, S.D. C, kymograph of TPX2-Halo and TPX2-710-Halo on microtubules. Vertical scale bar (time), 60 s; horizontal scale bar, 2 μm.

FIGURE 3.

Characterization of Eg5 in mammalian cell extracts. A, Western blot of cell extract and purified Eg5. B, schematic diagram of the single molecule TIRF experiments (left) and TIRF images of Eg5-EGFP accumulating at the microtubule plus-end (right). C, kymographs of kinesin-1 EGFP dimers and Eg5-EGFP from extracts on the same microtubule. Note the different time scale. Plus- and minus-ends of the microtubules are indicated. D, histogram of Eg5-EGFP motor velocity. E, histogram of the fluorescence of kinesin-1 dimers (light gray) and Eg5 molecules (dark gray) in the extract. F, schematic diagram (left) and fluorescence images (right) showing microtubule-microtubule sliding by Eg5. The arrowhead marks the end of the sliding microtubule. G, Coomassie Brilliant Blue-stained gel of Eg5-EGFP purified from insect cells and the trace of absorbance at 488 nm on the size exclusion column for the purified protein. The Western blot shown is for the fractions obtained from size exclusion chromatography of Eg5-mEmerald from LLC-Pk1 extract probed for Eg5. H, quantification of the velocity of Eg5-EGFP after the addition of DMSO, STLC, or FCPT (right). Error bars, S.E. I, directional and diffusive motility of Eg5-EGFP in the presence of 0, 20, or 50 mm KCl added to the motility buffer. Top, kymographs; bottom, mean squared displacement. Horizontal scale bar (B, C, F, and I), 1 μm; vertical scale bar (I), 60 s. Vertical scale in C is shown on the image. A.U., arbitrary units.

FIGURE 4.

Inhibition of Eg5 by TPX2 requires both binding to the microtubule and an interaction between TPX2 and Eg5. A, kymographs of Eg5-EGFP before and following the addition of TPX2 or TPX2–710; arrowhead, time of the TPX2 addition. B, quantification of Eg5-EGFP velocity; error bars, S.D. C, kymograph of kinesin-1 EGFP dimers walking on microtubules before and after the addition of TPX2 (arrowhead). 1 nm kinesin-1 EGFP (green) and 500 nm TPX2-Halo (red) were used. D, kymographs of Eg5-EGFP (green) before and following the addition of 20 nm TPX2-Halo (red). Right panels, enlarged view. E, kymographs of Eg5-EGFP that was premixed with TPX2-Halo or TPX2–710-Halo. F, quantification of Eg5-EGFP velocity in the presence of 50 nm TPX2 that was Halo-tagged (left) or untagged (right). Error bars, S.E. Horizontal scale bars (A, C, and E), 1 μm; horizontal scale bar (D), 2 μm; vertical scale bar, 60 s (A, D, and E) and 5 s (C).

FIGURE 5.

Differential regulation of Eg5 dimers, but not monomers, by full-length and truncated TPX2. Shown is velocity of microtubule gliding driven by Eg5 dimers (A) or Eg5 monomers (B). Error bars, S.E. C, model for inhibition of Eg5 by TPX2. Top, inhibition of motor stepping by full-length (left, stop symbol) and truncated TPX2 (right, slow symbol) in single molecule assays. Bottom panels, inhibition of microtubule gliding by Eg5 dimers (top) and Eg5 monomers (bottom). Green, Eg5; orange, TPX2.