Figure 6. Kinetochore recruitment of Mad2 is insufficient for SAC signal propagation in Mps1 mutant neuroblasts. (A) Top panel: Images showing the GFP-Mad2 fluorescent signals in Mps1 (ald^B4–2) mutant neuroblasts under normal mitotic progression. The arrowhead in image 1 indicates nuclear localized GFP-Mad2 at prophase. The arrows in the dashed-line circled regions in images 7 and 8 show the beginning of daughter cell formation. No kinetochore GFP-Mad2 fluorescent signals could be detected throughout the cycle. Middle panel: Images showing the GFP-Mad2 fluorescent signals in Mps1 (ald^B4–2) mutant neuroblasts treated with 5 μM colchicine. The arrowhead in image 9 shows the nuclear localized GFP-Mad2 at prophase. The arrows in images 11–14 show that a certain amount of the GFP-Mad2 protein can still be recruited to the kinetochores during the time course. The arrowhead in image 16 shows that the GFP-Mad2 fluorescent signal reappears in the newly formed nucleus to indicate the mitotic progression has bypassed the SAC arrest. Bottom panel: Images showing the GFP-Mad2 fluorescent signals in wild-type neuroblasts treated with 5 μM colchicine. Cells are arrested with strong and persistent kinetochore accumulated GFP-Mad2 fluorescent signals (arrows) during the time course. The arrowhead in image 17 indicates the nuclear localized GFP-Mad2 in prophase. Bar = 2 μm. Time, minutes, seconds. (B) Spinning disk confocal images displaying the region of interests for the peak levels of the GFP-Cdc20 on the kinetochores in neuroblasts after treatment with 5 μM colchicine (region a, d and e) and the relevant cytoplasmic regions (b, c and f) used as the background subtraction. The images were taken from either the wild-type (gfp-mad2; ald⁺, left); the ald^B4–2 heterozygotes (gfp-mad2; +/ald^B4–2, middle) or the Mps1 mutant (gfp-mad2; ald^B4–2, right). Bar = 3 μm. (C) The graph shows the quantified results measured from “b.” Approximately 20.2 ± 7.4% and 79.8 ± 2.42% of the wild-type levels of GFP-Mad2 can still be recruited to the kinetochores in the neuroblasts of the Mps1 mutant (ald^B4–2) and heterozygous background. Twenty individual images from each cell line were used for this quantification. (D) Western blot results showing relevant Mps1 protein expression levels in wild-type, heterozygous and homozygous of the ald^B4–2 third instar larvae brain samples. Actin bands act as the loading control.