Figure 2. TPX2 levels correlate with RanGTP and Eg5 dependence and spindle size. (A) Representative Western blot of dilution series of X. tropicalis and X. laevis egg extracts showing higher levels of TPX2 present in X. tropicalis. β-Tubulin is shown as a loading control. TPX2 is ∼82 kD, but migrates on a gel at ∼100 kD. Band intensities from Western blots of three extracts each were quantified (Odyssey software), and on average TPX2 levels were approximately threefold higher in X. tropicalis compared with X. laevis, giving an estimated concentration of 300 nM (X. laevis TPX2 concentration is ∼100 nM; Gruss et al., 2001). (B, left) Addition of 200 nM MBP-TPX2 to X. laevis extracts decreased spindle size. Polar localization of recombinant protein is detected by immunofluorescence against the MBP tag. Bar, 10 µm. (right) Quantification of spindle length with addition of 200 nM MBP-TPX2 compared with control addition of 200 nM MBP. Mean ± SD; n ≥ 671 spindles in each condition from three separate extracts; ***, P < 0.0001 from unpaired t test. (C) Quantification of spindle bipolarity as an indicator of proper spindle formation upon inhibition of the RanGTP pathway in X. laevis and X. tropicalis egg extracts. For each condition, all MT structures near condensed DNA were scored for morphology with n ≥ 100 for each extract in three separate experiments. Percentage of bipolarity was calculated from total structures scored. Mean ± SD. Dark gray bars indicate control extracts with no treatment. With 5 µM RanT24N addition, spindle formation in X. laevis egg extracts was impaired and the fraction of bipolar structures decreased to 7.3% (left red bar) with the remaining structures monopolar (39%) or lacking MTs (45.8%). When X. laevis extracts were supplemented with 200 nM MBP-TPX2, spindle bipolarity with RanT24N treatment was strongly rescued (left blue bar). TPX2 addition alone did not affect bipolarity (left light gray bar). X. tropicalis only showed a modest decrease with RanT24N treatment, from 54 to 46% (right blue bar). However, when X. tropicalis was immunodepleted of TPX2, bipolarity was almost completely lost upon RanT24N treatment, which reduced bipolar spindle assembly to 2.7% (right red bar). TPX2 immunodepletion from X. tropicalis extracts did not affect spindle bipolarity (right light gray bar). (D) Quantification of spindle bipolarity of X. laevis, X. laevis + 200 nM MBP-TPX2, and X. tropicalis MT structures upon monastrol treatment at the indicated concentration. MT structures were scored as in C but normalized to the 0-µM condition for each extract and plotted as the mean ± standard error. Four-parameter logistic fit is shown for both X. tropicalis (blue dashed line; IC50 of 211 µM) and X. laevis (red dashed line; IC50 of 21 µM), but no fit could be calculated for the X. laevis + TPX2 condition as it was resistant to monastrol treatment at the highest concentration.
Figure 2.

TPX2 levels correlate with RanGTP and Eg5 dependence and spindle size. (A) Representative Western blot of dilution series of X. tropicalis and X. laevis egg extracts showing higher levels of TPX2 present in X. tropicalis. β-Tubulin is shown as a loading control. TPX2 is ∼82 kD, but migrates on a gel at ∼100 kD. Band intensities from Western blots of three extracts each were quantified (Odyssey software), and on average TPX2 levels were approximately threefold higher in X. tropicalis compared with X. laevis, giving an estimated concentration of 300 nM (X. laevis TPX2 concentration is ∼100 nM; Gruss et al., 2001). (B, left) Addition of 200 nM MBP-TPX2 to X. laevis extracts decreased spindle size. Polar localization of recombinant protein is detected by immunofluorescence against the MBP tag. Bar, 10 µm. (right) Quantification of spindle length with addition of 200 nM MBP-TPX2 compared with control addition of 200 nM MBP. Mean ± SD; n ≥ 671 spindles in each condition from three separate extracts; ***, P < 0.0001 from unpaired t test. (C) Quantification of spindle bipolarity as an indicator of proper spindle formation upon inhibition of the RanGTP pathway in X. laevis and X. tropicalis egg extracts. For each condition, all MT structures near condensed DNA were scored for morphology with n ≥ 100 for each extract in three separate experiments. Percentage of bipolarity was calculated from total structures scored. Mean ± SD. Dark gray bars indicate control extracts with no treatment. With 5 µM RanT24N addition, spindle formation in X. laevis egg extracts was impaired and the fraction of bipolar structures decreased to 7.3% (left red bar) with the remaining structures monopolar (39%) or lacking MTs (45.8%). When X. laevis extracts were supplemented with 200 nM MBP-TPX2, spindle bipolarity with RanT24N treatment was strongly rescued (left blue bar). TPX2 addition alone did not affect bipolarity (left light gray bar). X. tropicalis only showed a modest decrease with RanT24N treatment, from 54 to 46% (right blue bar). However, when X. tropicalis was immunodepleted of TPX2, bipolarity was almost completely lost upon RanT24N treatment, which reduced bipolar spindle assembly to 2.7% (right red bar). TPX2 immunodepletion from X. tropicalis extracts did not affect spindle bipolarity (right light gray bar). (D) Quantification of spindle bipolarity of X. laevis, X. laevis + 200 nM MBP-TPX2, and X. tropicalis MT structures upon monastrol treatment at the indicated concentration. MT structures were scored as in C but normalized to the 0-µM condition for each extract and plotted as the mean ± standard error. Four-parameter logistic fit is shown for both X. tropicalis (blue dashed line; IC50 of 211 µM) and X. laevis (red dashed line; IC50 of 21 µM), but no fit could be calculated for the X. laevis + TPX2 condition as it was resistant to monastrol treatment at the highest concentration.

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