Prometaphase spindle dynamics in Drosophila embryos: steady-state and elongation phases. (A, top) Time series of a spindle in living transgenic Drosophila embryos expressing GFP-tubulin (green) and histone-RFP (red) taken at the indicated time points on the plot of pole–pole spacing from the same spindle (red arrows on inset of bottom panel; t = 0 is NEB). (bottom) Averaged and normalized pole–pole dynamics in cycle 11 Drosophila embryo spindles; t = 0 is NEB. Prometaphase dynamics (blue): the steady-state (I) and elongation (II) phases are shown. Error bars show SD. (B) The top two rows show KLP61F-GFP (first row) and Ncd-GFP (second row) localization during prometaphase (phase I; steady-state) in restored spindles (deconvolved 3D image stacks), respectively. The left panels are 3D views of restored spindles, and the right panels show a single z plane from the same spindles. The bottom two rows show the colocalization of KLP61F-GFP and Ncd-GFP with rhodamine-tubulin on the nascent ipMTs during prometaphase (phase I; steady-state). (C, top) Averaged and normalized prometaphase pole–pole dynamics from cycle 11 embryos in wild type, control IgG, Ncd null, and KLP61F antibody–injected wild-type (black) and Ncd null (red). Error bars show SD. (middle) Data from the top panel are shown without SDs for clarity. (bottom) Pole–pole dynamics in individual representative spindles. (D) Cartoon of phase I (left) and II (right) prometaphase spindles based on in vivo functional perturbation and localization data. Colored arrows below the drawings indicate the direction of the forces generated by corresponding motors. Bars, 5 μm.