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First published online May 24, 2006
doi: 10.1242/10.1242/jcs.02957

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Antagonistic activities of Klp10A and Orbit regulate spindle length, bipolarity and function in vivo

Cancer Research UK Cell Cycle Genetics Group, University of Cambridge, Department of Genetics, Cambridge, CB2 3EH, UK

View larger version (25K): [in a new window]   Fig. 1. RNAi of Klp10A diminishes the degree of apoptosis associated with Orbit depletion. (A) Knockdown of orbit alone (pink line) decreases the number of 2N cells while slightly increasing the number of presumptive mitotic cells with a 4N DNA content and dramatically increasing the number found in apoptosis relative to controls (solid green). (B) Klp10A downregulation (blue line) reduces the number of 2N cells while lowering and broadening the 4N peak, suggesting aneuploidy. (C) Double RNAi of orbit and Klp10A (black line) decreases the number of apoptotic cells seen following orbit RNAi as well as slightly increasing the proportion of 2N cells. Also shown are the relative distributions of cell population following each of the indicated treatments. (D) Western blot analysis of cell extracts taken from dsRNA-treated cells reveals substantial protein knockdown 72 hours after treatment. Actin is the loading control.

View larger version (55K): [in a new window]   Fig. 2. Bipolar spindle morphology is restored in cells co-depleted of Orbit and Klp10A. (A-D") Cells were treated with the indicated dsRNAs and stained for the distribution of microtubules (-tubulin; red), -tubulin (green) and DNA (blue) 72 hours later. (A,A') Control metaphase spindles are bipolar and bi-centrosomal with a centrosome (green) attached to each end. (B) orbit RNAi commonly results in monopolar spindles with both centrosomes at the centre of a monoaster. Bipolar spindles may form in Orbit depleted cells, although these tend to be abnormally short (B'). After Klp10A knockdown, spindles form that are on average about 150% the length of controls. Half of these spindles are marked by the presence of both centrosomes at a single, well focused, spindle pole and an acentrosomal pole that is focused to varying degrees (C,C'). Double RNAi against orbit and Klp10A rescues bipolar spindle morphology. These bipolar spindles can be placed in three classes: bipolar bi-centrosomal spindles morphologically similar to controls (D), bipolar bi-centrosomal spindles significantly longer than controls (D') and bipolar monoastral spindles similar in length to controls but which have an asymmetric localisation of -tubulin. In this cell, the -tubulin diffusely stains one pole while at the other it forms a large aggregate in the centre of the aster, suggesting the presence of both centrosomes (D", see text for details). (E) Distribution of spindle lengths following each of the indicated treatments. Note the enhancement of spindle length in Klp10A single downregulated cells and the shortening of those in cells depleted of Orbit. The double knockdown of orbit and Klp10A can rescue the average spindle length and further restores the distribution of spindle lengths to values similar to that seen in the controls. All images are shown at the same magnification. Bars, 10 µm.

View larger version (35K): [in a new window]   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).

View larger version (46K): [in a new window]   Fig. 4. Time-lapse imaging reveals that simultaneous RNAi of orbit and Klp10A rescues spindle bipolarity by preventing spindle collapse. (A,B) Selected frames from time-lapse sequences of cells depleted of both Orbit and Klp10A. Time is in seconds relative to prometaphase onset. Unlike in cells downregulated for either Orbit or Klp10A alone, the separation distance between the centrosomes at prophase remains constant or increases during spindle formation. Note the large variation in time needed for anaphase onset between the double-knockdown cells. See text for details. (C) Kinetic profiles of centrosome separation for the cells shown in Fig. 3 and Fig. 4A. Prometaphase onset occurs at 0 seconds. Bars, 10 µm.

View larger version (45K): [in a new window]   Fig. 5. Double orbit- and Klp10A-depleted cells have reduced intra-centromeric tension and retain BubR1 at their kinetochores. S2 cells were treated with dsRNA for 72 hours before being fixed and stained to localise microtubules (-tubulin; red), DNA (blue) and, in green, either CID (A) or BubR1 (C). (A) Chromosome configurations representing the different classes measured are shown. (B) Distributions of intra-centromeric distances under each of the indicated conditions. Following bi-orientation in control cells, metaphase centromeres are placed under tension as revealed by the increased intra-centromeric distances relative to unattached `relaxed' prophase centromeres. Knockdown of Klp10A and orbit, either singly or simultaneously, alters the degree of centromeric separation. Note how the distributions and magnitudes of the separation distances increase when spindles are capped on both ends by an aster, compared with bipolar spindles with an aster at only one end. Even when placed on bi-astral, bipolar spindles, the centromeres in double-knockdown cells are under less tension than those in controls. See text for details. (C) BubR1 localisation correlates with spindle morphology and chromosome position. In control metaphase cells, BubR1 is seen as a few faint punctae. By contrast, in orbit knockdown cells that form monopolar spindles, the staining intensity at kinetochores is markedly increased, consistent with a failure to bi-orient and lack of tension. Downregulation of Klp10A does not prevent bipolar spindle formation, chromosome bi-orientation or congression. As with controls, equatorially positioned chromosomes in these cells show little BubR1 staining, whereas those that are lagging at the spindle pole and are presumably mono-oriented exhibit a robust signal. Equatorially positioned chromosomes in orbit and Klp10A double-RNAi cells display intense BubR1 staining on some kinetochores but not others. Bars, 5 µm.