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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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Cyclin B degradation is delayed in a SAC-independent manner in aurA NBs.(a–d) (Far left column panels) RFP-histone H2A was used to monitor chromosome dynamics (red in the top panels and in the middle monochrome panels), and cyclin B-GFP was used to determine the amounts of cyclin B protein in individual cells (green and lower monochrome panels). The panels of the middle column show the cyclin B-GFP degradation profiles of the corresponding genotypes. The timing of mitosis (far right column panels) is indicated for each individual NB. The black and grey bars indicate the durations of prometaphase and metaphase, respectively. Time (h:min:s) is indicated at the top left of each image. NEBD began at 00:00:00. Scale bars, 5 μm. (a) Timing of mitosis and cyclin B-GFP degradation in WT NBs (n=18). (b) Timing of mitosis and cyclin B-GFP degradation in aurA14641/aurA17961 NBs (n=23). (c) Timing of mitosis and cyclin B-GFP degradation in aurA mad2 double-mutant NBs (n=17). (d) Timing of mitosis and cyclin B-GFP degradation in aurA  mutant NBs (n=12). (e) Graph showing the mean time (±s.d.) to 50% cyclin B-GFP degradation for WT, mad2, aurA mad2 and aurA NBs. WT: 4.8±1.1 min (n=18); AurA14641/aurA17961: 10.3±3.4 min (n=23); aurA: 46.2±12.6 (n=12); and aurA mad2: 23.3±8.9 min (n=17). ***P<7 × 10−5 (Wilcoxon test).
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f4: Cyclin B degradation is delayed in a SAC-independent manner in aurA NBs.(a–d) (Far left column panels) RFP-histone H2A was used to monitor chromosome dynamics (red in the top panels and in the middle monochrome panels), and cyclin B-GFP was used to determine the amounts of cyclin B protein in individual cells (green and lower monochrome panels). The panels of the middle column show the cyclin B-GFP degradation profiles of the corresponding genotypes. The timing of mitosis (far right column panels) is indicated for each individual NB. The black and grey bars indicate the durations of prometaphase and metaphase, respectively. Time (h:min:s) is indicated at the top left of each image. NEBD began at 00:00:00. Scale bars, 5 μm. (a) Timing of mitosis and cyclin B-GFP degradation in WT NBs (n=18). (b) Timing of mitosis and cyclin B-GFP degradation in aurA14641/aurA17961 NBs (n=23). (c) Timing of mitosis and cyclin B-GFP degradation in aurA mad2 double-mutant NBs (n=17). (d) Timing of mitosis and cyclin B-GFP degradation in aurA mutant NBs (n=12). (e) Graph showing the mean time (±s.d.) to 50% cyclin B-GFP degradation for WT, mad2, aurA mad2 and aurA NBs. WT: 4.8±1.1 min (n=18); AurA14641/aurA17961: 10.3±3.4 min (n=23); aurA: 46.2±12.6 (n=12); and aurA mad2: 23.3±8.9 min (n=17). ***P<7 × 10−5 (Wilcoxon test).

Mentions: Anaphase onset is directly connected to anaphase promoting complex/cyclosome (APC/C) activation and the degradation of mitotic targets, including cyclin B and securin, therefore we monitored cyclin B-GFP degradation in aurA and aurA mad2 mutants. Consistent with previous findings, cyclin B-GFP accumulated at the centrosome and in the nucleus of WT NBs before entry into mitosis. Following NEBD, cyclin B-GFP was visible throughout the cell, but the fluorescence was particularly intense in the kinetochore region and mitotic spindle1921 (Fig. 4a and Supplementary Movie 3). The cyclin B-GFP signal began to disappear abruptly, shortly after the last chromosome reached the metaphase plate, and half the cyclin B-GFP present was degraded 4.75±1.04 min (n=18) after metaphase plate formation (Fig. 4e). The cells of the aurA14641/aurA17961mutant took 11.3±3.5 min (n=23) to degrade half their cyclin B (Fig. 4b,e and Supplementary Movie 4), whereas aurA mitotic cells took 46.2±12.6 min (n=12; Fig. 4d,e and Supplementary Movie 6). The introduction of the mad2 mutation in the aurA mutant shortened the time required for half the cyclin B to be degraded to 23.2±8.9 min (n=17) but did not restore the WT timing of mitosis (Fig. 4c,e and Supplementary Movie 5). Consistent with observations in fixed anaphase specimens, live imaging of aurA and aurA mad2 mutant cells showed no lagging/delayed chromatids during anaphase that might have resulted from incorrect attachment1622. This suggests that the machinery responsible for correcting inappropriate attachments between kinetochores and spindle microtubules is fully functional. As cells with Aurora A defects display delayed mitosis, we investigated whether AurA overexpression could accelerate mitosis, as in some cancer cell lines23. However, we found that mitotic timing was normal under normal conditions in NBs overexpressing Aurora A kinase, with no acceleration (Supplementary Fig. 4). Furthermore, these NBs remained arrested in M phase in the presence of microtubule depolymerizing drugs (unpublished observations).


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)

Cyclin B degradation is delayed in a SAC-independent manner in aurA NBs.(a–d) (Far left column panels) RFP-histone H2A was used to monitor chromosome dynamics (red in the top panels and in the middle monochrome panels), and cyclin B-GFP was used to determine the amounts of cyclin B protein in individual cells (green and lower monochrome panels). The panels of the middle column show the cyclin B-GFP degradation profiles of the corresponding genotypes. The timing of mitosis (far right column panels) is indicated for each individual NB. The black and grey bars indicate the durations of prometaphase and metaphase, respectively. Time (h:min:s) is indicated at the top left of each image. NEBD began at 00:00:00. Scale bars, 5 μm. (a) Timing of mitosis and cyclin B-GFP degradation in WT NBs (n=18). (b) Timing of mitosis and cyclin B-GFP degradation in aurA14641/aurA17961 NBs (n=23). (c) Timing of mitosis and cyclin B-GFP degradation in aurA mad2 double-mutant NBs (n=17). (d) Timing of mitosis and cyclin B-GFP degradation in aurA  mutant NBs (n=12). (e) Graph showing the mean time (±s.d.) to 50% cyclin B-GFP degradation for WT, mad2, aurA mad2 and aurA NBs. WT: 4.8±1.1 min (n=18); AurA14641/aurA17961: 10.3±3.4 min (n=23); aurA: 46.2±12.6 (n=12); and aurA mad2: 23.3±8.9 min (n=17). ***P<7 × 10−5 (Wilcoxon test).
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f4: Cyclin B degradation is delayed in a SAC-independent manner in aurA NBs.(a–d) (Far left column panels) RFP-histone H2A was used to monitor chromosome dynamics (red in the top panels and in the middle monochrome panels), and cyclin B-GFP was used to determine the amounts of cyclin B protein in individual cells (green and lower monochrome panels). The panels of the middle column show the cyclin B-GFP degradation profiles of the corresponding genotypes. The timing of mitosis (far right column panels) is indicated for each individual NB. The black and grey bars indicate the durations of prometaphase and metaphase, respectively. Time (h:min:s) is indicated at the top left of each image. NEBD began at 00:00:00. Scale bars, 5 μm. (a) Timing of mitosis and cyclin B-GFP degradation in WT NBs (n=18). (b) Timing of mitosis and cyclin B-GFP degradation in aurA14641/aurA17961 NBs (n=23). (c) Timing of mitosis and cyclin B-GFP degradation in aurA mad2 double-mutant NBs (n=17). (d) Timing of mitosis and cyclin B-GFP degradation in aurA mutant NBs (n=12). (e) Graph showing the mean time (±s.d.) to 50% cyclin B-GFP degradation for WT, mad2, aurA mad2 and aurA NBs. WT: 4.8±1.1 min (n=18); AurA14641/aurA17961: 10.3±3.4 min (n=23); aurA: 46.2±12.6 (n=12); and aurA mad2: 23.3±8.9 min (n=17). ***P<7 × 10−5 (Wilcoxon test).
Mentions: Anaphase onset is directly connected to anaphase promoting complex/cyclosome (APC/C) activation and the degradation of mitotic targets, including cyclin B and securin, therefore we monitored cyclin B-GFP degradation in aurA and aurA mad2 mutants. Consistent with previous findings, cyclin B-GFP accumulated at the centrosome and in the nucleus of WT NBs before entry into mitosis. Following NEBD, cyclin B-GFP was visible throughout the cell, but the fluorescence was particularly intense in the kinetochore region and mitotic spindle1921 (Fig. 4a and Supplementary Movie 3). The cyclin B-GFP signal began to disappear abruptly, shortly after the last chromosome reached the metaphase plate, and half the cyclin B-GFP present was degraded 4.75±1.04 min (n=18) after metaphase plate formation (Fig. 4e). The cells of the aurA14641/aurA17961mutant took 11.3±3.5 min (n=23) to degrade half their cyclin B (Fig. 4b,e and Supplementary Movie 4), whereas aurA mitotic cells took 46.2±12.6 min (n=12; Fig. 4d,e and Supplementary Movie 6). The introduction of the mad2 mutation in the aurA mutant shortened the time required for half the cyclin B to be degraded to 23.2±8.9 min (n=17) but did not restore the WT timing of mitosis (Fig. 4c,e and Supplementary Movie 5). Consistent with observations in fixed anaphase specimens, live imaging of aurA and aurA mad2 mutant cells showed no lagging/delayed chromatids during anaphase that might have resulted from incorrect attachment1622. This suggests that the machinery responsible for correcting inappropriate attachments between kinetochores and spindle microtubules is fully functional. As cells with Aurora A defects display delayed mitosis, we investigated whether AurA overexpression could accelerate mitosis, as in some cancer cell lines23. However, we found that mitotic timing was normal under normal conditions in NBs overexpressing Aurora A kinase, with no acceleration (Supplementary Fig. 4). Furthermore, these NBs remained arrested in M phase in the presence of microtubule depolymerizing drugs (unpublished observations).

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