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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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Mitotic delay in aurA mutants is SAC independent.(a) Analysis of mitotic indices (±s.d.) in WT, mad2, aurA and aurA mad2 neural tissues. WT: 0.73±0.69% (n=11,332, three brains); mad2: 0.96±1.07% (n=9,507, three brains); aurA: 6.7±2.1% (n=11,334, three brains); and aurA mad2: 5.8±2.8% (n=8,648, three brains). ***P<10−10 (Wilcoxon test). (b) Chromosome morphology in WT, mad2, aurA and aurA mad2 neural tissues. Note that in both aurA (bottom left) and aurA mad2 (bottom right) mutants, the mitotic cells have highly condensed chromosomes, indicative of a delay in mitosis, unlike WT and mad2 mutant brain cells (top). Scale bar, 10 μm. (c) Western blots showing the levels of cyclin B, AurA, Mad2, phospho-histone H3 (Ser10) (left) and cyclin A (right). Actin was used as a loading control. Cyclin B and phospho-histone H3 (Ser10) are high in aurA and aurA mad2 neural tissue extracts, whereas cyclin A protein levels are not. The truncated AurAK377/STOP corresponding to the aurA8839 mutation is detected as a higher mobility protein in the corresponding aurA and aurA mad2 brain extracts (red triangle), whereas the endogenous Aurora A is indicated by the blue triangle. (d) Cyclin A degradation occurs during prometaphase in WT, mad2, aurA and aurA mad2 mitotic cells. WT and mutant brains of the indicated genotypes were fixed and stained for cyclin A (green and lower monochrome panels) during G2 and prometaphase. Microtubules are labelled in red (and on the middle monochrome panels) and phospho-histone H3 (Ser10) is displayed in blue. (e) Cyclin B degradation occurs at the metaphase to anaphase transition in WT, mad2, aurA and aurA mad2 mutant brains. Brains were fixed and stained for cyclin B (green and lower monochrome panels), tubulin (red and middle monochrome panels) and phospho-histone H3 (Ser10). Scale bar, 10 μm.
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f2: Mitotic delay in aurA mutants is SAC independent.(a) Analysis of mitotic indices (±s.d.) in WT, mad2, aurA and aurA mad2 neural tissues. WT: 0.73±0.69% (n=11,332, three brains); mad2: 0.96±1.07% (n=9,507, three brains); aurA: 6.7±2.1% (n=11,334, three brains); and aurA mad2: 5.8±2.8% (n=8,648, three brains). ***P<10−10 (Wilcoxon test). (b) Chromosome morphology in WT, mad2, aurA and aurA mad2 neural tissues. Note that in both aurA (bottom left) and aurA mad2 (bottom right) mutants, the mitotic cells have highly condensed chromosomes, indicative of a delay in mitosis, unlike WT and mad2 mutant brain cells (top). Scale bar, 10 μm. (c) Western blots showing the levels of cyclin B, AurA, Mad2, phospho-histone H3 (Ser10) (left) and cyclin A (right). Actin was used as a loading control. Cyclin B and phospho-histone H3 (Ser10) are high in aurA and aurA mad2 neural tissue extracts, whereas cyclin A protein levels are not. The truncated AurAK377/STOP corresponding to the aurA8839 mutation is detected as a higher mobility protein in the corresponding aurA and aurA mad2 brain extracts (red triangle), whereas the endogenous Aurora A is indicated by the blue triangle. (d) Cyclin A degradation occurs during prometaphase in WT, mad2, aurA and aurA mad2 mitotic cells. WT and mutant brains of the indicated genotypes were fixed and stained for cyclin A (green and lower monochrome panels) during G2 and prometaphase. Microtubules are labelled in red (and on the middle monochrome panels) and phospho-histone H3 (Ser10) is displayed in blue. (e) Cyclin B degradation occurs at the metaphase to anaphase transition in WT, mad2, aurA and aurA mad2 mutant brains. Brains were fixed and stained for cyclin B (green and lower monochrome panels), tubulin (red and middle monochrome panels) and phospho-histone H3 (Ser10). Scale bar, 10 μm.

Mentions: We therefore created various double-mutant lines for aurA8839 and mad2, and checked the double mutation by confirming that the Mad2 protein was absent and that there was a higher mobility form of the truncated AurAK377/stop mutant protein (Fig. 2c, second panel from the top). We confirmed that the mad2P mutant (hereafter referred to as mad2) was viable414. In mad2 larval brains, the number of NBs (±s.d.) (73.2±9.0 NBs per lobe, n=22) was similar to that in the wild type (75.4±10.5 NBs per lobe, n=14) (Fig. 1a left panels and b).


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)

Mitotic delay in aurA mutants is SAC independent.(a) Analysis of mitotic indices (±s.d.) in WT, mad2, aurA and aurA mad2 neural tissues. WT: 0.73±0.69% (n=11,332, three brains); mad2: 0.96±1.07% (n=9,507, three brains); aurA: 6.7±2.1% (n=11,334, three brains); and aurA mad2: 5.8±2.8% (n=8,648, three brains). ***P<10−10 (Wilcoxon test). (b) Chromosome morphology in WT, mad2, aurA and aurA mad2 neural tissues. Note that in both aurA (bottom left) and aurA mad2 (bottom right) mutants, the mitotic cells have highly condensed chromosomes, indicative of a delay in mitosis, unlike WT and mad2 mutant brain cells (top). Scale bar, 10 μm. (c) Western blots showing the levels of cyclin B, AurA, Mad2, phospho-histone H3 (Ser10) (left) and cyclin A (right). Actin was used as a loading control. Cyclin B and phospho-histone H3 (Ser10) are high in aurA and aurA mad2 neural tissue extracts, whereas cyclin A protein levels are not. The truncated AurAK377/STOP corresponding to the aurA8839 mutation is detected as a higher mobility protein in the corresponding aurA and aurA mad2 brain extracts (red triangle), whereas the endogenous Aurora A is indicated by the blue triangle. (d) Cyclin A degradation occurs during prometaphase in WT, mad2, aurA and aurA mad2 mitotic cells. WT and mutant brains of the indicated genotypes were fixed and stained for cyclin A (green and lower monochrome panels) during G2 and prometaphase. Microtubules are labelled in red (and on the middle monochrome panels) and phospho-histone H3 (Ser10) is displayed in blue. (e) Cyclin B degradation occurs at the metaphase to anaphase transition in WT, mad2, aurA and aurA mad2 mutant brains. Brains were fixed and stained for cyclin B (green and lower monochrome panels), tubulin (red and middle monochrome panels) and phospho-histone H3 (Ser10). Scale bar, 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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f2: Mitotic delay in aurA mutants is SAC independent.(a) Analysis of mitotic indices (±s.d.) in WT, mad2, aurA and aurA mad2 neural tissues. WT: 0.73±0.69% (n=11,332, three brains); mad2: 0.96±1.07% (n=9,507, three brains); aurA: 6.7±2.1% (n=11,334, three brains); and aurA mad2: 5.8±2.8% (n=8,648, three brains). ***P<10−10 (Wilcoxon test). (b) Chromosome morphology in WT, mad2, aurA and aurA mad2 neural tissues. Note that in both aurA (bottom left) and aurA mad2 (bottom right) mutants, the mitotic cells have highly condensed chromosomes, indicative of a delay in mitosis, unlike WT and mad2 mutant brain cells (top). Scale bar, 10 μm. (c) Western blots showing the levels of cyclin B, AurA, Mad2, phospho-histone H3 (Ser10) (left) and cyclin A (right). Actin was used as a loading control. Cyclin B and phospho-histone H3 (Ser10) are high in aurA and aurA mad2 neural tissue extracts, whereas cyclin A protein levels are not. The truncated AurAK377/STOP corresponding to the aurA8839 mutation is detected as a higher mobility protein in the corresponding aurA and aurA mad2 brain extracts (red triangle), whereas the endogenous Aurora A is indicated by the blue triangle. (d) Cyclin A degradation occurs during prometaphase in WT, mad2, aurA and aurA mad2 mitotic cells. WT and mutant brains of the indicated genotypes were fixed and stained for cyclin A (green and lower monochrome panels) during G2 and prometaphase. Microtubules are labelled in red (and on the middle monochrome panels) and phospho-histone H3 (Ser10) is displayed in blue. (e) Cyclin B degradation occurs at the metaphase to anaphase transition in WT, mad2, aurA and aurA mad2 mutant brains. Brains were fixed and stained for cyclin B (green and lower monochrome panels), tubulin (red and middle monochrome panels) and phospho-histone H3 (Ser10). Scale bar, 10 μm.
Mentions: We therefore created various double-mutant lines for aurA8839 and mad2, and checked the double mutation by confirming that the Mad2 protein was absent and that there was a higher mobility form of the truncated AurAK377/stop mutant protein (Fig. 2c, second panel from the top). We confirmed that the mad2P mutant (hereafter referred to as mad2) was viable414. In mad2 larval brains, the number of NBs (±s.d.) (73.2±9.0 NBs per lobe, n=22) was similar to that in the wild type (75.4±10.5 NBs per lobe, n=14) (Fig. 1a left panels and b).

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