Fig. 7. The N-terminal domain of pEg2 inhibits bipolar spindle assembly and destabilises previously assembled bipolar spindles in Xenopus egg extract. The bipolar mitotic spindle assembly assay was performed as previously described (Roghi et al., 1998). After fixation, the spindles were scored under a fluorescence microscope. (A) Bipolar spindle (top) and monopolar spindle (bottom). The spindle incorporates rhodamine-labelled tubulin and appears in red. DNA is stained by Hoechst dye (blue). (B) 400 ng/µl (final concentration) of either the Nt-pEg2-(His)6 protein or a control pMAL peptide were added during spindle assembly. In the presence of Nt-pEg2-(His)6, only 34±18% (3 different experiments) of the spindles remained bipolar instead of 81±12% (4 different experiments) in the control. (C) Bipolar spindles previously assembled in Xenopus egg extract were incubated for 1 hour with 400 ng/µl of Nt-pEg2(His)6 protein, or with the control pMAL peptide at the same concentration. In the presence of Nt-pEg2-(His)6, 55±5% (3 different experiments) of the spindles remained bipolar instead of 82±8% in the control (4 different experiments).