Kinetochore localized Mad2 and Cdc20 is itself insufficient for triggering the mitotic checkpoint when Mps1 is low in Drosophila melanogaster neuroblasts

Abstract

The relationships between the kinetochore and checkpoint control remain unresolved. Here, we report the characterization of the in vivo behavior of Cdc20 and Mad2 and the relevant spindle assembly checkpoint (SAC) functions in the neuroblasts of a Drosophila Mps1 weak allele (ald (B4-2) ). ald (B4-2) third instar larvae brain samples contain only around 16% endogenous Mps1 protein, and the SAC function is abolished. However, this does not lead to rapid anaphase onset and mitotic exit, in contrast to the loss of Mad2 alone in a mad2 (EY) mutant. The level of GFP-Cdc20 recruitment to the kinetochore is unaffected in ald (B4-2) neuroblasts, while the level of GFP-Mad2 is reduced to just about 20%. Cdc20 and Mad2 display only monophasic exponential kinetics at the kinetochores. The ald (B4-2) heterozygotes expressed approximately 65% of normal Mps1 protein levels, and this is enough to restore the SAC function. The kinetochore recruitment of GFP-Mad2 in response to SAC activation increases by around 80% in heterozygotes, compared with just about 20% in ald (B4-2) mutant. This suggests a correlation between Mps1 levels and Mad2 kinetochore localization and perhaps the existence of a threshold level at which Mps1 is fully functional. The failure to arrest the mitotic progression in ald (B4-2) neuroblasts in response to colchicine treatment suggests that when Mps1 levels are low, approximately 20% of normal GFP-Mad2, alongside normal levels of GFP-Cdc20 kinetochore recruitments, is insufficient for triggering SAC signal propagation.

Figure 1. Western blot results showing that the Mps1 weak allele ald^B4–2 retains approximately 16.4% of the endogenous Mps1 protein. (A) Western blot results showing samples prepared from the third instar larvae brains for w⁶⁷, ald^B4–2, gfp-cdc20; ald^B4–2 and gfp-mad2; ald^B4–2 lines. A small amount of the Mps1 endogenous protein in samples from ald^B4–2, gfp-cdc20; ald^B4–2 and gfp-mad2; ald^B4–2 lines can be still detected. Expression of GFP-Cdc20 (panel 2) and GFP-Mad2 (panel 3) fusion proteins has been confirmed in the relevant ald^B4–2 genetic background as indicated. The actin bands are used as a loading control. All the antibodies were used in 1:500 dilutions. The western blot bands were imaged using an Odyssey machine. (B) Quantification results showing the western blot band intensities for Mps1 in relation to the above samples after normalization with the intensity of the relevant actin band. The data are presented as a percentage of the Mps1 in the wild-type (w⁶⁷) sample. The intensity for each sample was quantified from three independent western blots using TINA software.

Figure 2. SAC in ald^B4–2 neuroblasts is defective, and this can be rescued by introducing an ectopically expressed GFP-Mps1 fusion protein. Third instar larvae prepared from wild-type (gfp-cdc20; ald⁺, top panel), an Mps1 mutant (gfp-cdc20; ald^B4–2, middle panel) and a rescued Mps1 mutant line (gfp-mps1; ald^B4–2, bottom panel) were treated with 5 μM colchicine. GFP-Cdc20 behavior was then examined in the neuroblasts. The GFP-Cdc20 fluorescent signal accumulates predominantly in the neuroblast cytoplasm in interphase (arrowheads in all panels at 00:00 time point). It is strong and persistently associated with arrested kinetochores after colchicine treatment (top panel, arrows) in wild-type neuroblasts. In contrast, the GFP-Cdc20 signal continues to oscillate on and off the kinetochores (middle panel, arrows), and the protein was transported to the cytoplasm, when the cell enters interphase so that the daughter nucleus reappears as a dark shape (arrowheads in the middle panel at 00:00 and 35:00 min time points). This suggests that the ald^B4–2 neuroblast mitosis has bypassed the SAC arrest. This defective SAC can be restored by introducing a copy of the gfp-mps1 fusion transgene. The kinetochores with strong and persistent GFP-Mps1 fluorescent signals reappear (bottom panel, arrows), suggesting the cell cycle is arrested (bottom panel). The images were scanned using a spinning disk confocal at 22°C with 488 nm laser excitation. Time, minutes, seconds. Bar = 2 μm.

Figure 3. Comparison of the mitotic progression in neuroblasts using GFP-Cdc20 as marker. GFP-Cdc20 was excluded from the interphase (Int.) nucleus (images 1 and 11, arrowheads), entering the nucleus by early prophase (Pro.) (Images 2 and 12, arrowhead). GFP-Cdc20 could be readily observed on prophase and prometaphase kinetochores (Images 3, 4, 13 and14, arrowheads) and persisted on metaphase (Met.) (Images 5 and 15, arrowheads) and greatly declined from anaphase kinetochores (Images 6 and 16, arrowheads). The beginning of cytokinesis immediately after telophase (Telo.) is marked by the arrowhead at image 18, when the cell begins to elongate and bud. Interphase in the daughter cell is marked by the beginning of the reappearance of the dark nucleus as the GFP-Cdc20 relocated to the cytoplasm (Image 20, arrowhead) the chromatin morphologies showing relevant cell cycle stages were determined using coexpressed His2B-mRFP as a marker (Images 21–30, arrows). The entire neuroblast was encompassed in the dashed-line circled region. Time-lapse images were recorded using a spinning disk confocal system at 22°C. Bar = 2 μm. Please refer to Table 1 for the actual timing of mitotic progression.

Figure 4. The level of kinetochore recruitment of Cdc20 is not affected by reduction of the Mps1 in ald^B4–2 mutant. (A) Spinning disk confocal time-lapse images show GFP-Cdc20 localization patterns in third instar larvae neuroblasts from wild-type (gfp-cdc20; ald⁺, top panel) or Mps1 mutant (gfp-cdc20; ald^B4–2, bottom panel). Arrows indicate kinetochore-associated GFP-Cdc20 signals. Arrowheads indicate the prophase nucleus just before NEB (nuclear envelope breakdown, Image at 00:00 time point) or the early interphase nucleus (Image at 26:00 time point) where GFP-Cdc20 was excluded from the nucleus. Bar = 2μm. (B) GFP-Cdc20 fluorescent intensities at prometaphase kinetochores (selected regions a and c) were quantified and compared after subtraction of the relevant cytoplasmic backgrounds (selected regions b and d). Spinning disk confocal images displaying the peak levels of the GFP-Cdc20 at the prometaphase kinetochores were taken from either the wild-type (gfp-cdc20; ald⁺, left) or the Mps1 mutant (gfp-cdc20; ald^B4–2, right). Bar = 3 μm. (C) The graph shows the quantification results. There is no significant different between the highest intensities of the GFP-Cdc20 recruited at prometaphase kinetochores in Mps1 mutant (gfp-cdc20; ald^B4–2) and in the wild-type (gfp-cdc20; ald⁺) neuroblasts. Twenty individual neuroblast images from each fly line were used for this quantification. Time, minutes, seconds.

Figure 5. The absence of Mad2 causes slower Cdc20 dynamics at kinetochores. (A) An example of the FRAP experiments performed on the GFP-Cdc20 signals from a group of prometaphase kinetochores (arrows) in neuroblasts from the gfp-cdc20; ald⁺ wild-type line. Image A1 displays the GFP-Cdc20 fluorescent intensity on the prometaphase kinetochores before photobleaching. Image A2 shows that the GFP-Cdc20 fluorescent intensity at kinetochores in the region of interest “a” was reduced to levels equivalent to that of “b” in cytoplasm after photobleaching. Images A3–7 are selected images from the time-lapse movies showing the GFP-Cdc20 fluorescent intensity recoveries on kinetochores monitored over 2 min post bleach. The cytoplasmic area of the neuroblast is encompassed within the dashed circle in each picture. (B) An example of the normalized GFP-Cdc20 fluorescence intensity recovery time-lapse curve (blue dots) in neuroblasts from the wild-type background (gfp-cdc20; ald⁺ genotype). The red line represents the best-fitted single exponential curve for the GFP-Cdc20 analyzed by fitting nonlinear regression curves using GraphPad Prism. The original fluorescent intensity level before bleaching has been indicated by an arrow. The value representing the half-life recovery (t_1/2) has been indicated by the double arrowheads. (C) An example of the normalized GFP-Cdc20 fluorescent intensity recovery time-lapse curve (green dots) in neuroblasts from Mps1 (ald^B4–2) mutant background (gfp-cdc20; ald^B4–2). The red line shows the best-fitted single exponential curve. (D) FRAP analysis results representing the half-life fluorescent intensity recoveries at prometaphase or colchicine arrested metaphase kinetochores post-photo bleach. The asterisks indicate the p values showing significant differences between the compared pairs. The numbers of the experiments used in these assays were indicated in Table 2.

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.