Fig. 1. Mutations in bub3 result in premature sister chromatid separation, abnormal anaphase and aneuploidy. (A) Western blot of total protein extracts from wild-type (Oregon R), bub3¹ homozygous and bub3¹/Df(3R)Dr-rv1 neuroblasts with anti-Bub3 antibody. -tubulin was detected as a loading control. (B) Immunolocalization of Bub3 in wild-type and bub3¹ mutant neuroblasts (n=354) showing that the Bub3 mutant protein fails to localize to kinetochores in 46% of the mitotic cells. In 45% of the prometaphases of bub3¹ mutant cells, Bub3 can localize to some kinetochores, whereas in only 9% of prometaphases is Bub3 detected with reduced intensity at all kinetochores. Polo was used to label kinetochores. DNA is shown in blue, polo in red and bub3 in green in the merged image. (C) Mitotic index of third instar larvae neuroblasts from Oregon R, bub3¹/bub3¹ and bub3¹/Df(3R)Dr-rv1, after a 1 hour incubation in colchicine. bub3¹ mutant cells fail to sustain mitotic arrest upon spindle damage. (D) Squashed preparations of wild-type and bub3¹ neuroblasts after incubation in colchicine. Upon spindle damage, bub3¹ mutant cells fail to maintain sister chromatid cohesion. (E) Squashed preparations of wild-type and bub3¹ mutant larvae neuroblasts at different stages of mitosis. bub3¹ mutant neuroblasts show aneuploidy, PSCS and anaphases with lagging chromatids. (F) Quantification of mitotic parameters in Oregon R, bub3¹ homozygous and bub3¹/Df(3R)Dr-rv1 third instar larvae neuroblasts, showing that homozygous and hemizygous cells behave differently during the initial stages of mitosis. Bar, 5 µm.