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Aurora-A kinase is required for centrosome maturation in Caenorhabditis elegans.

Hannak E, Kirkham M, Hyman AA, Oegema K - J. Cell Biol. (2001)

Bottom Line: Consistent with this hypothesis, we find that AIR-1 is required for the increase in centrosomal gamma-tubulin and two other PCM components, ZYG-9 and CeGrip, as embryos enter mitosis.Furthermore, the AIR-1-dependent increase in centrosomal gamma-tubulin does not require MTs.These results suggest that aurora-A kinases are required to execute a MT-independent pathway for the recruitment of PCM during centrosome maturation.

View Article: PubMed Central - PubMed

Affiliation: Max Planck Institute of Molecular Cell Biology and Genetics, 01307 Dresden, Germany.

ABSTRACT
Centrosomes mature as cells enter mitosis, accumulating gamma-tubulin and other pericentriolar material (PCM) components. This occurs concomitant with an increase in the number of centrosomally organized microtubules (MTs). Here, we use RNA-mediated interference (RNAi) to examine the role of the aurora-A kinase, AIR-1, during centrosome maturation in Caenorhabditis elegans. In air-1(RNAi) embryos, centrosomes separate normally, an event that occurs before maturation in C. elegans. After nuclear envelope breakdown, the separated centrosomes collapse together, and spindle assembly fails. In mitotic air-1(RNAi) embryos, centrosomal alpha-tubulin fluorescence intensity accumulates to only 40% of wild-type levels, suggesting a defect in the maturation process. Consistent with this hypothesis, we find that AIR-1 is required for the increase in centrosomal gamma-tubulin and two other PCM components, ZYG-9 and CeGrip, as embryos enter mitosis. Furthermore, the AIR-1-dependent increase in centrosomal gamma-tubulin does not require MTs. These results suggest that aurora-A kinases are required to execute a MT-independent pathway for the recruitment of PCM during centrosome maturation.

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The effect of AIR-1 depletion on the accumulation of centrosomal γ-tubulin is MT independent. Wild-type (left) and air-1(RNAi) embryos (right) were treated with nocodazole, fixed, and stained for MTs, DNA, γ-tubulin, and AIR-1. γ-Tubulin staining is reduced dramatically, and two small foci of γ-tubulin staining are observed for each centrosome in air-1(RNAi) embryos (compare wild-type and air-1(RNAi) insets). Insets are magnified 5.5-fold. Bar, 10 μm.
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fig5: The effect of AIR-1 depletion on the accumulation of centrosomal γ-tubulin is MT independent. Wild-type (left) and air-1(RNAi) embryos (right) were treated with nocodazole, fixed, and stained for MTs, DNA, γ-tubulin, and AIR-1. γ-Tubulin staining is reduced dramatically, and two small foci of γ-tubulin staining are observed for each centrosome in air-1(RNAi) embryos (compare wild-type and air-1(RNAi) insets). Insets are magnified 5.5-fold. Bar, 10 μm.

Mentions: Our results show that AIR-1 is required for the increase in centrosomally organized MTs and for the accumulation of centrosomal γ-tubulin as embryos enter mitosis. To test the possibility that the decrease in centrosomally organized MTs is responsible for the failure to accumulate centrosomal γ-tubulin, we treated wild-type and air-1(RNAi) embryos with nocodazole to depolymerize MTs and stained them for MTs, DNA, γ-tubulin, and AIR-1 (Fig. 5). MTs were completely depolymerized in nocodazole-treated embryos with only centrosomes detectable using an α-tubulin antibody (Fig. 5, top). Embryos were treated with nocodazole for 7 min before fixation (see Materials and methods). Since pronuclear migration requires MTs, we analyzed embryos with condensed chromosomes in which the pronuclei were still separated. This separation indicates that MT depolymerization occurred at a stage similar to the −256 s panel in Fig. 3 A, which is before the accumulation of additional centrosomal γ-tubulin. Despite complete depolymerization of MTs, centrosomes in wild-type embryos accumulated γ-tubulin and AIR-1 to normal levels during the 7 min incubation in nocodazole. In contrast, centrosomes in nocodazole-treated air-1(RNAi) embryos remained small, similar to those in untreated air-1(RNAi) embryos. From these results, we conclude that the AIR-1–dependent accumulation of centrosomal γ-tubulin during maturation does not require MTs.


Aurora-A kinase is required for centrosome maturation in Caenorhabditis elegans.

Hannak E, Kirkham M, Hyman AA, Oegema K - J. Cell Biol. (2001)

The effect of AIR-1 depletion on the accumulation of centrosomal γ-tubulin is MT independent. Wild-type (left) and air-1(RNAi) embryos (right) were treated with nocodazole, fixed, and stained for MTs, DNA, γ-tubulin, and AIR-1. γ-Tubulin staining is reduced dramatically, and two small foci of γ-tubulin staining are observed for each centrosome in air-1(RNAi) embryos (compare wild-type and air-1(RNAi) insets). Insets are magnified 5.5-fold. Bar, 10 μm.
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Related In: Results  -  Collection

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fig5: The effect of AIR-1 depletion on the accumulation of centrosomal γ-tubulin is MT independent. Wild-type (left) and air-1(RNAi) embryos (right) were treated with nocodazole, fixed, and stained for MTs, DNA, γ-tubulin, and AIR-1. γ-Tubulin staining is reduced dramatically, and two small foci of γ-tubulin staining are observed for each centrosome in air-1(RNAi) embryos (compare wild-type and air-1(RNAi) insets). Insets are magnified 5.5-fold. Bar, 10 μm.
Mentions: Our results show that AIR-1 is required for the increase in centrosomally organized MTs and for the accumulation of centrosomal γ-tubulin as embryos enter mitosis. To test the possibility that the decrease in centrosomally organized MTs is responsible for the failure to accumulate centrosomal γ-tubulin, we treated wild-type and air-1(RNAi) embryos with nocodazole to depolymerize MTs and stained them for MTs, DNA, γ-tubulin, and AIR-1 (Fig. 5). MTs were completely depolymerized in nocodazole-treated embryos with only centrosomes detectable using an α-tubulin antibody (Fig. 5, top). Embryos were treated with nocodazole for 7 min before fixation (see Materials and methods). Since pronuclear migration requires MTs, we analyzed embryos with condensed chromosomes in which the pronuclei were still separated. This separation indicates that MT depolymerization occurred at a stage similar to the −256 s panel in Fig. 3 A, which is before the accumulation of additional centrosomal γ-tubulin. Despite complete depolymerization of MTs, centrosomes in wild-type embryos accumulated γ-tubulin and AIR-1 to normal levels during the 7 min incubation in nocodazole. In contrast, centrosomes in nocodazole-treated air-1(RNAi) embryos remained small, similar to those in untreated air-1(RNAi) embryos. From these results, we conclude that the AIR-1–dependent accumulation of centrosomal γ-tubulin during maturation does not require MTs.

Bottom Line: Consistent with this hypothesis, we find that AIR-1 is required for the increase in centrosomal gamma-tubulin and two other PCM components, ZYG-9 and CeGrip, as embryos enter mitosis.Furthermore, the AIR-1-dependent increase in centrosomal gamma-tubulin does not require MTs.These results suggest that aurora-A kinases are required to execute a MT-independent pathway for the recruitment of PCM during centrosome maturation.

View Article: PubMed Central - PubMed

Affiliation: Max Planck Institute of Molecular Cell Biology and Genetics, 01307 Dresden, Germany.

ABSTRACT
Centrosomes mature as cells enter mitosis, accumulating gamma-tubulin and other pericentriolar material (PCM) components. This occurs concomitant with an increase in the number of centrosomally organized microtubules (MTs). Here, we use RNA-mediated interference (RNAi) to examine the role of the aurora-A kinase, AIR-1, during centrosome maturation in Caenorhabditis elegans. In air-1(RNAi) embryos, centrosomes separate normally, an event that occurs before maturation in C. elegans. After nuclear envelope breakdown, the separated centrosomes collapse together, and spindle assembly fails. In mitotic air-1(RNAi) embryos, centrosomal alpha-tubulin fluorescence intensity accumulates to only 40% of wild-type levels, suggesting a defect in the maturation process. Consistent with this hypothesis, we find that AIR-1 is required for the increase in centrosomal gamma-tubulin and two other PCM components, ZYG-9 and CeGrip, as embryos enter mitosis. Furthermore, the AIR-1-dependent increase in centrosomal gamma-tubulin does not require MTs. These results suggest that aurora-A kinases are required to execute a MT-independent pathway for the recruitment of PCM during centrosome maturation.

Show MeSH