X. tropicalis and X. laevis spindles differ in morphology and sensitivity to inhibition of Ran and Eg5. (A) Spinning disk confocal images of X. laevis and X. tropicalis spindle midzones in live spindle reactions on polyethylene glycol–coated glass. Bar, 5 µm. Mean line scan intensity of MTs across the length of the spindle (as described in Materials and methods), showing reduced MT density in the center of the spindle of X. tropicalis; n = 15 spindles for X. laevis and n = 19 for X. tropicalis from three extracts. (B) Inhibition of the RanGTP pathway with 1 µM of the dominant-negative mutant RanT24N disrupted spindle assembly in X. laevis but not X. tropicalis egg extracts. Bar, 10 µm. (C) Line scan quantification of TPX2 immunofluorescence relative to tubulin intensity from >50 spindles in each condition from one representative experiment. TPX2 intensity was higher in X. tropicalis spindles (dark blue) and remained unchanged upon addition of 1 µM RanT24N (light blue), whereas X. laevis spindles recruited less TPX2 (red), which was lost upon RanT24N treatment (pink). Although spindle assembly was strongly inhibited in X. laevis upon RanT24N treatment, the remaining bipolar MT structures formed in these reactions were used for line scan analysis. (D) Representative images of spindle assembly reactions in X. laevis and X. tropicalis egg extracts in the presence of increasing amounts of monastrol to inhibit Eg5. X. laevis spindles began to shorten and collapse with increasing monastrol, whereas X. tropicalis spindles showed little collapse and were resistant to monastrol except at the highest concentrations. Bar, 10 µm.