Figure 2.

The N-terminal domain of MCAK is required for efficient spindle assembly. CSF extracts were depleted of endogenous MCAK (MΔ) or mock-depleted with IgG (IgGΔ), cycled into interphase with CaCl₂, and cycled back into mitosis with additional depleted CSF extract. GFP-tagged proteins were added back to 100 nM final concentration with the second addition of CSF extract. Spindles were assembled for 90 min, fixed, and sedimented onto coverslips, and the chromatin was stained with Hoechst and mounted with anti-fade. (A) Images of representative structures from the add-back experiments are shown with MTs in magenta and chromatin in green. Scale bar, 20 μm. (B) Quantification of the spindles, large asters, and monopolar structures observed in the extracts that are represented in A. The data are graphed as the mean percentage plus the SEM from four independent extracts. An asterisk indicates a significant difference relative to GMCAK add-back.