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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.
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