Figure 4. TPX2 regulates spindle size and MT distribution through interaction with Eg5. (A) Addition of TPX2 domain truncation mutants to X. laevis extract. The TPX2ΔAurA mutant localized like full-length TPX2 and had similar effects on spindle size, whereas TPX2ΔEg5 did not localize to the spindle or cause a decrease in spindle length. Bar, 10 µm. Mean ± SD; n ≥ 166 spindles in each condition from three separate extracts preserved by squashing under coverslips with spindle fixative. ΔEg5 TPX2 mean spindle length was not significantly different from the MBP control, but was significantly different from full-length TPX2 by unpaired t test; ***, P < 0.0001. (B) Representative images of Eg5 localization and MT density changes in X. laevis spindles upon addition of 200 nM MBP-TPX2 and in comparison to X. tropicalis. Left panels are merged, middle panels show rhodamine-tubulin, and right panels show Eg5 immunofluorescence. Bar, 10 µm. (C) Comparison of phenotypes in X. laevis extracts with the addition of either 200 nM MBP-TPX2 or 50 µM monastrol to inhibit Eg5. Although both treatments reduced spindle length, the insets demonstrate a significant decrease in MT density in the center of the spindle with TPX2 addition but not monastrol treatment. Bars, 10 µm. (D) Line scan quantification of Eg5 immunofluorescence intensity in X. tropicalis and X. laevis spindles with and without addition of TPX2 mutants (see Materials and methods). Spindle lengths were normalized for statistical analysis. Mean ± standard error; n ≥ 169 spindles for each condition from three extracts. (E) Line scan quantification of MT density measured by rhodamine-tubulin intensity in X. tropicalis and X. laevis spindles with and without addition of TPX2 (see Materials and methods). Spindle lengths were normalized for statistical analysis. Mean ± standard error; n ≥ 156 spindles for each condition from three extracts.
Figure 4.

TPX2 regulates spindle size and MT distribution through interaction with Eg5. (A) Addition of TPX2 domain truncation mutants to X. laevis extract. The TPX2ΔAurA mutant localized like full-length TPX2 and had similar effects on spindle size, whereas TPX2ΔEg5 did not localize to the spindle or cause a decrease in spindle length. Bar, 10 µm. Mean ± SD; n ≥ 166 spindles in each condition from three separate extracts preserved by squashing under coverslips with spindle fixative. ΔEg5 TPX2 mean spindle length was not significantly different from the MBP control, but was significantly different from full-length TPX2 by unpaired t test; ***, P < 0.0001. (B) Representative images of Eg5 localization and MT density changes in X. laevis spindles upon addition of 200 nM MBP-TPX2 and in comparison to X. tropicalis. Left panels are merged, middle panels show rhodamine-tubulin, and right panels show Eg5 immunofluorescence. Bar, 10 µm. (C) Comparison of phenotypes in X. laevis extracts with the addition of either 200 nM MBP-TPX2 or 50 µM monastrol to inhibit Eg5. Although both treatments reduced spindle length, the insets demonstrate a significant decrease in MT density in the center of the spindle with TPX2 addition but not monastrol treatment. Bars, 10 µm. (D) Line scan quantification of Eg5 immunofluorescence intensity in X. tropicalis and X. laevis spindles with and without addition of TPX2 mutants (see Materials and methods). Spindle lengths were normalized for statistical analysis. Mean ± standard error; n ≥ 169 spindles for each condition from three extracts. (E) Line scan quantification of MT density measured by rhodamine-tubulin intensity in X. tropicalis and X. laevis spindles with and without addition of TPX2 (see Materials and methods). Spindle lengths were normalized for statistical analysis. Mean ± standard error; n ≥ 156 spindles for each condition from three extracts.

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