Fig. 3. Spindle formation in living S2 cells expressing GFP-tubulin after orbit or Klp10A RNAi. Selected frames from time-lapse sequences showing spindle formation and mitotic progression in (A) control, (B) orbit RNAi- and (C) Klp10A RNAi-treated cells. (A) In control cells the two separated centrosomes oppose one another on the nuclear envelope during prophase. At prometaphase onset (0 s) the nuclear envelope becomes fenestrated and astral MTs interact with the kinetochores to form a bipolar spindle (100) that becomes more robust as the chromosomes congress to the equatorially positioned metaphase plate (400), the fluorescence `shadow' at the equator indicates the presence of the chromosomes. The spindle in this cell slightly shortens at metaphase (1480) after assuming a steady state length which it maintains until anaphase onset (2180) (see also supplementary material Movie 1). (B) orbit RNAi does not prevent prophase centrosome separation. As the chromosomes become bi-oriented during prometaphase, the nascent bipolar spindle collapses upon itself (0-630 seconds) to form a monopolar spindle (900). In this cell, transient multi-poles (1260-1770 seconds; arrowheads) form that are probably generated by individual or small clusters of chromosomes as evidenced by the shadow at their equators (see text for details). (C) Knockdown of Klp10A does not perturb the initial separation of the centrosomes at prophase but, as illustrated here, causes their subsequent collapse during prometaphase in 50% of the cells followed by time-lapse microscopy (0-520 s). Unlike Orbit-depleted cells, those lacking Klp10A are able to form stable bipolar spindles (1400-1700 seconds) that are monoastral. These spindles are fully functional and cells can enter into anaphase (3220). Time (in brackets) is in seconds relative to prometaphase onset. Bars, 10 µm (see also supplementary material Movie 2).