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Spindle assembly checkpoint inactivation fails to suppress neuroblast tumour formation in aurA mutant Drosophila.

Caous R, Pascal A, Romé P, Richard-Parpaillon L, Karess R, Giet R - Nat Commun (2015)

Bottom Line: By contrast, disrupting the SAC in the aurA mutant does not prevent NB amplification, tumour formation or chromosome segregation.Thus, the NBs of aurA mutants present delayed mitosis, with accurate chromosome segregation occurring in a SAC-independent manner.We report here the existence of an Aurora A-dependent mechanism promoting efficient, timed cyclin B degradation.

View Article: PubMed Central - PubMed

Affiliation: Institut de Génétique et Développement de Rennes-Université de Rennes I-CNRS- UMR 6290, 2 avenue du Pr Léon Bernard, 35043 Rennes, France.

ABSTRACT
Tissue homeostasis requires accurate control of cell proliferation, differentiation and chromosome segregation. Drosophila sas-4 and aurA mutants present brain tumours with extra neuroblasts (NBs), defective mitotic spindle assembly and delayed mitosis due to activation of the spindle assembly checkpoint (SAC). Here we inactivate the SAC in aurA and sas-4 mutants to determine whether the generation of aneuploidy compromises NB proliferation. Inactivation of the SAC in the sas-4 mutant impairs NB proliferation and disrupts euploidy. By contrast, disrupting the SAC in the aurA mutant does not prevent NB amplification, tumour formation or chromosome segregation. The monitoring of Mad2 and cyclin B dynamics in live aurA NBs reveals that SAC satisfaction is not coupled to cyclin B degradation. Thus, the NBs of aurA mutants present delayed mitosis, with accurate chromosome segregation occurring in a SAC-independent manner. We report here the existence of an Aurora A-dependent mechanism promoting efficient, timed cyclin B degradation.

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Metaphase to anaphase transition is delayed in aurA hypomorphic mutant NBs.(a) Mitotic timing and spindle assembly checkpoint analysis in 26 WT (left) and 22 aurA14641/aurA17961NBs (right). The grey and white bars indicate the time spent by each cell in prometaphase and metaphase, respectively. The green bar indicates the time for which GFP-Mad2 was present at the kinetochores. (b) Box plots showing prometaphase duration (left, WT: 3.27±0.93 min; aurA14641/aurA17961: 8.81±3.42 min; P=1.2 × 10−11, Wilcoxon test), metaphase duration (middle, WT: 3.7±1.3 min; aurA14641/aurA17961: 11.6±3.8 min; P=1.0 × 10−11, Wilcoxon test) and Mad2 occupancy time at the kinetochore (right, WT: 5.2±1.1 min; aurA14641/aurA17961: 14.6±5.4 min; P=3.0x10−13, Wilcoxon test) in WT (n=54) and aurA14641/aurA17961 (n=22) NBs. The above values are the means±s.d. Box plot: boxes show the upper and lower quartile. P values are determined with a Wilcoxon test. (c) Box plots showing the time interval between SAC satisfaction and mitotic entry. In WT cells, the time between SAC satisfaction and anaphase onset was 1.76±0.94 min (n=54). By contrast, this interval was much longer, and significantly so, in the aurA mutant, at 5.75±1.88 min (n=22). ***P=1.9 × 10−10, Wilcoxon test. Box plot: boxes show the upper and lower quartile. P values are determined with a Wilcoxon test. (d,e) RFP-histone H2A was used to monitor chromosome dynamics (red in the top panels and in the monochrome middle panels), and GFP-Mad2 (green and bottom lower monochrome panels) was used to monitor chromosome attachment and SAC satisfaction. Scale bars, 10 μm. Time (min:s) is indicated at the top left of each image. NEBD began at 00:00. (d) Analysis of chromosome and GFP-Mad2 dynamics in WT NBs. (e) Analysis of chromosome and GFP-Mad2 dynamics in aurA14641/aurA17961 NBs.
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f3: Metaphase to anaphase transition is delayed in aurA hypomorphic mutant NBs.(a) Mitotic timing and spindle assembly checkpoint analysis in 26 WT (left) and 22 aurA14641/aurA17961NBs (right). The grey and white bars indicate the time spent by each cell in prometaphase and metaphase, respectively. The green bar indicates the time for which GFP-Mad2 was present at the kinetochores. (b) Box plots showing prometaphase duration (left, WT: 3.27±0.93 min; aurA14641/aurA17961: 8.81±3.42 min; P=1.2 × 10−11, Wilcoxon test), metaphase duration (middle, WT: 3.7±1.3 min; aurA14641/aurA17961: 11.6±3.8 min; P=1.0 × 10−11, Wilcoxon test) and Mad2 occupancy time at the kinetochore (right, WT: 5.2±1.1 min; aurA14641/aurA17961: 14.6±5.4 min; P=3.0x10−13, Wilcoxon test) in WT (n=54) and aurA14641/aurA17961 (n=22) NBs. The above values are the means±s.d. Box plot: boxes show the upper and lower quartile. P values are determined with a Wilcoxon test. (c) Box plots showing the time interval between SAC satisfaction and mitotic entry. In WT cells, the time between SAC satisfaction and anaphase onset was 1.76±0.94 min (n=54). By contrast, this interval was much longer, and significantly so, in the aurA mutant, at 5.75±1.88 min (n=22). ***P=1.9 × 10−10, Wilcoxon test. Box plot: boxes show the upper and lower quartile. P values are determined with a Wilcoxon test. (d,e) RFP-histone H2A was used to monitor chromosome dynamics (red in the top panels and in the monochrome middle panels), and GFP-Mad2 (green and bottom lower monochrome panels) was used to monitor chromosome attachment and SAC satisfaction. Scale bars, 10 μm. Time (min:s) is indicated at the top left of each image. NEBD began at 00:00. (d) Analysis of chromosome and GFP-Mad2 dynamics in WT NBs. (e) Analysis of chromosome and GFP-Mad2 dynamics in aurA14641/aurA17961 NBs.

Mentions: We characterized this unexpected phenotype further by assessing SAC activation by following GFP-Mad2 recruitment and the timing of cyclin B degradation in live aurA mutant NBs. The mitotic delay was very long (∼60 min) for the aurA8839 allele. We therefore also used a combination of less severe alleles (aurA14641/aurA17961) (ref. 7), making it possible to minimize the bleaching of red fluorescent protein (RFP)-Histone H2A and GFP-tagged proteins during the time course of the experiments (Fig. 3a,b). In WT NBs, GFP-Mad2 labelled the nuclear envelope and nucleoplasm before mitosis, and weak fluorescence was observed at the kinetochores during prometaphase (Fig. 3d and Supplementary Movie 1), consistent with previous findings1920. In all WT cells examined, this weak GFP-Mad2 signal at the kinetochores completely disappeared 1.76±0.85 min (n=55) before anaphase onset (Fig. 3c). In aurA14641/aurA17961 mutant NBs, GFP-Mad2 strongly labelled the kinetochores, consistent with defective kinetochore–microtubule attachment and strong SAC activation (Fig. 3a,b,e and Supplementary Movie 2). This strong GFP-Mad2 kinetochore signal decreased slowly in intensity as the chromosomes congressed to the metaphase plate, indicating a delay in the establishment of correct kinetochore–microtubule attachments (Fig. 3e, top and Supplementary Movie 2). However, the time lag between the disappearance of GFP-Mad2 from the kinetophores and the onset of anaphase was significantly longer (5.75±1.88 min, n=22) in all dividing aurA14641/aurA17961 cells than in WT cells (Fig. 3c).


Spindle assembly checkpoint inactivation fails to suppress neuroblast tumour formation in aurA mutant Drosophila.

Caous R, Pascal A, Romé P, Richard-Parpaillon L, Karess R, Giet R - Nat Commun (2015)

Metaphase to anaphase transition is delayed in aurA hypomorphic mutant NBs.(a) Mitotic timing and spindle assembly checkpoint analysis in 26 WT (left) and 22 aurA14641/aurA17961NBs (right). The grey and white bars indicate the time spent by each cell in prometaphase and metaphase, respectively. The green bar indicates the time for which GFP-Mad2 was present at the kinetochores. (b) Box plots showing prometaphase duration (left, WT: 3.27±0.93 min; aurA14641/aurA17961: 8.81±3.42 min; P=1.2 × 10−11, Wilcoxon test), metaphase duration (middle, WT: 3.7±1.3 min; aurA14641/aurA17961: 11.6±3.8 min; P=1.0 × 10−11, Wilcoxon test) and Mad2 occupancy time at the kinetochore (right, WT: 5.2±1.1 min; aurA14641/aurA17961: 14.6±5.4 min; P=3.0x10−13, Wilcoxon test) in WT (n=54) and aurA14641/aurA17961 (n=22) NBs. The above values are the means±s.d. Box plot: boxes show the upper and lower quartile. P values are determined with a Wilcoxon test. (c) Box plots showing the time interval between SAC satisfaction and mitotic entry. In WT cells, the time between SAC satisfaction and anaphase onset was 1.76±0.94 min (n=54). By contrast, this interval was much longer, and significantly so, in the aurA mutant, at 5.75±1.88 min (n=22). ***P=1.9 × 10−10, Wilcoxon test. Box plot: boxes show the upper and lower quartile. P values are determined with a Wilcoxon test. (d,e) RFP-histone H2A was used to monitor chromosome dynamics (red in the top panels and in the monochrome middle panels), and GFP-Mad2 (green and bottom lower monochrome panels) was used to monitor chromosome attachment and SAC satisfaction. Scale bars, 10 μm. Time (min:s) is indicated at the top left of each image. NEBD began at 00:00. (d) Analysis of chromosome and GFP-Mad2 dynamics in WT NBs. (e) Analysis of chromosome and GFP-Mad2 dynamics in aurA14641/aurA17961 NBs.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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f3: Metaphase to anaphase transition is delayed in aurA hypomorphic mutant NBs.(a) Mitotic timing and spindle assembly checkpoint analysis in 26 WT (left) and 22 aurA14641/aurA17961NBs (right). The grey and white bars indicate the time spent by each cell in prometaphase and metaphase, respectively. The green bar indicates the time for which GFP-Mad2 was present at the kinetochores. (b) Box plots showing prometaphase duration (left, WT: 3.27±0.93 min; aurA14641/aurA17961: 8.81±3.42 min; P=1.2 × 10−11, Wilcoxon test), metaphase duration (middle, WT: 3.7±1.3 min; aurA14641/aurA17961: 11.6±3.8 min; P=1.0 × 10−11, Wilcoxon test) and Mad2 occupancy time at the kinetochore (right, WT: 5.2±1.1 min; aurA14641/aurA17961: 14.6±5.4 min; P=3.0x10−13, Wilcoxon test) in WT (n=54) and aurA14641/aurA17961 (n=22) NBs. The above values are the means±s.d. Box plot: boxes show the upper and lower quartile. P values are determined with a Wilcoxon test. (c) Box plots showing the time interval between SAC satisfaction and mitotic entry. In WT cells, the time between SAC satisfaction and anaphase onset was 1.76±0.94 min (n=54). By contrast, this interval was much longer, and significantly so, in the aurA mutant, at 5.75±1.88 min (n=22). ***P=1.9 × 10−10, Wilcoxon test. Box plot: boxes show the upper and lower quartile. P values are determined with a Wilcoxon test. (d,e) RFP-histone H2A was used to monitor chromosome dynamics (red in the top panels and in the monochrome middle panels), and GFP-Mad2 (green and bottom lower monochrome panels) was used to monitor chromosome attachment and SAC satisfaction. Scale bars, 10 μm. Time (min:s) is indicated at the top left of each image. NEBD began at 00:00. (d) Analysis of chromosome and GFP-Mad2 dynamics in WT NBs. (e) Analysis of chromosome and GFP-Mad2 dynamics in aurA14641/aurA17961 NBs.
Mentions: We characterized this unexpected phenotype further by assessing SAC activation by following GFP-Mad2 recruitment and the timing of cyclin B degradation in live aurA mutant NBs. The mitotic delay was very long (∼60 min) for the aurA8839 allele. We therefore also used a combination of less severe alleles (aurA14641/aurA17961) (ref. 7), making it possible to minimize the bleaching of red fluorescent protein (RFP)-Histone H2A and GFP-tagged proteins during the time course of the experiments (Fig. 3a,b). In WT NBs, GFP-Mad2 labelled the nuclear envelope and nucleoplasm before mitosis, and weak fluorescence was observed at the kinetochores during prometaphase (Fig. 3d and Supplementary Movie 1), consistent with previous findings1920. In all WT cells examined, this weak GFP-Mad2 signal at the kinetochores completely disappeared 1.76±0.85 min (n=55) before anaphase onset (Fig. 3c). In aurA14641/aurA17961 mutant NBs, GFP-Mad2 strongly labelled the kinetochores, consistent with defective kinetochore–microtubule attachment and strong SAC activation (Fig. 3a,b,e and Supplementary Movie 2). This strong GFP-Mad2 kinetochore signal decreased slowly in intensity as the chromosomes congressed to the metaphase plate, indicating a delay in the establishment of correct kinetochore–microtubule attachments (Fig. 3e, top and Supplementary Movie 2). However, the time lag between the disappearance of GFP-Mad2 from the kinetophores and the onset of anaphase was significantly longer (5.75±1.88 min, n=22) in all dividing aurA14641/aurA17961 cells than in WT cells (Fig. 3c).

Bottom Line: By contrast, disrupting the SAC in the aurA mutant does not prevent NB amplification, tumour formation or chromosome segregation.Thus, the NBs of aurA mutants present delayed mitosis, with accurate chromosome segregation occurring in a SAC-independent manner.We report here the existence of an Aurora A-dependent mechanism promoting efficient, timed cyclin B degradation.

View Article: PubMed Central - PubMed

Affiliation: Institut de Génétique et Développement de Rennes-Université de Rennes I-CNRS- UMR 6290, 2 avenue du Pr Léon Bernard, 35043 Rennes, France.

ABSTRACT
Tissue homeostasis requires accurate control of cell proliferation, differentiation and chromosome segregation. Drosophila sas-4 and aurA mutants present brain tumours with extra neuroblasts (NBs), defective mitotic spindle assembly and delayed mitosis due to activation of the spindle assembly checkpoint (SAC). Here we inactivate the SAC in aurA and sas-4 mutants to determine whether the generation of aneuploidy compromises NB proliferation. Inactivation of the SAC in the sas-4 mutant impairs NB proliferation and disrupts euploidy. By contrast, disrupting the SAC in the aurA mutant does not prevent NB amplification, tumour formation or chromosome segregation. The monitoring of Mad2 and cyclin B dynamics in live aurA NBs reveals that SAC satisfaction is not coupled to cyclin B degradation. Thus, the NBs of aurA mutants present delayed mitosis, with accurate chromosome segregation occurring in a SAC-independent manner. We report here the existence of an Aurora A-dependent mechanism promoting efficient, timed cyclin B degradation.

Show MeSH
Related in: MedlinePlus