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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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AIR-1 localizes to centrosomes and is required for spindle assembly. (A) The AIR-1 antibody detects a single band of ∼40 kD in extracts prepared from wild-type worms (left). (Right) Comparison of air-1(RNAi) worm extract with serial dilutions of wild-type extract (numbers indicate percentage of amount loaded in 100% lane) indicates >90% depletion of AIR-1. α-Tubulin was used as a loading control. (B) Wild-type embryos stained for MTs, DNA (left, green and red), and AIR-1 (right). A recently fertilized embryo (top) and a metaphase embryo (bottom) are shown. (C) A single deconvolved focal plane showing a centrosome from a metaphase embryo stained for γ-tubulin and AIR-1. (D) A mitotic air-1(RNAi) embryo stained for MTs, DNA (left, green and red), and AIR-1 (right). Bars: (B and D) 10 μm; (C) 2.5 μm.
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fig1: AIR-1 localizes to centrosomes and is required for spindle assembly. (A) The AIR-1 antibody detects a single band of ∼40 kD in extracts prepared from wild-type worms (left). (Right) Comparison of air-1(RNAi) worm extract with serial dilutions of wild-type extract (numbers indicate percentage of amount loaded in 100% lane) indicates >90% depletion of AIR-1. α-Tubulin was used as a loading control. (B) Wild-type embryos stained for MTs, DNA (left, green and red), and AIR-1 (right). A recently fertilized embryo (top) and a metaphase embryo (bottom) are shown. (C) A single deconvolved focal plane showing a centrosome from a metaphase embryo stained for γ-tubulin and AIR-1. (D) A mitotic air-1(RNAi) embryo stained for MTs, DNA (left, green and red), and AIR-1 (right). Bars: (B and D) 10 μm; (C) 2.5 μm.

Mentions: The C. elegans homologue of aurora-A, AIR-1, localizes to centrosomes and is required for normal spindle assembly (Schumacher et al., 1998). To investigate the role of aurora-A in the centrosome cycle, we first analyzed its localization during the first embryonic division. We raised an antibody to AIR-1 that detects a single band of ∼40 kD on Western blots. This band is reduced >90% in air-1(RNAi) worms (Fig. 1 A), confirming the specificity of our antibody. During fertilization, a sperm-derived centrosome enters the egg (which lacks centrosomes) and duplicates, resulting in two small centrosomes positioned between the sperm pronucleus and the cortex. AIR-1 localizes to centrosomes in early embryos (Fig. 1 B, top) and also weakly to astral and cytoplasmic MTs. In wild-type, the maternal pronucleus migrates toward the sperm pronucleus as the chromosomes condense. The pronuclei fuse, their nuclear envelopes break down, and the first mitotic spindle assembles. In mitotic embryos, AIR-1 concentrates in the centers of the asters (Fig. 1 B, bottom) in a donut-shaped region peripheral to γ-tubulin staining (Fig. 1 C) and also localizes along the base of astral MTs. The pattern of localization of AIR-1 is specific, since it is not observed in air-1(RNAi) embryos.


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

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

AIR-1 localizes to centrosomes and is required for spindle assembly. (A) The AIR-1 antibody detects a single band of ∼40 kD in extracts prepared from wild-type worms (left). (Right) Comparison of air-1(RNAi) worm extract with serial dilutions of wild-type extract (numbers indicate percentage of amount loaded in 100% lane) indicates >90% depletion of AIR-1. α-Tubulin was used as a loading control. (B) Wild-type embryos stained for MTs, DNA (left, green and red), and AIR-1 (right). A recently fertilized embryo (top) and a metaphase embryo (bottom) are shown. (C) A single deconvolved focal plane showing a centrosome from a metaphase embryo stained for γ-tubulin and AIR-1. (D) A mitotic air-1(RNAi) embryo stained for MTs, DNA (left, green and red), and AIR-1 (right). Bars: (B and D) 10 μm; (C) 2.5 μm.
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Related In: Results  -  Collection

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fig1: AIR-1 localizes to centrosomes and is required for spindle assembly. (A) The AIR-1 antibody detects a single band of ∼40 kD in extracts prepared from wild-type worms (left). (Right) Comparison of air-1(RNAi) worm extract with serial dilutions of wild-type extract (numbers indicate percentage of amount loaded in 100% lane) indicates >90% depletion of AIR-1. α-Tubulin was used as a loading control. (B) Wild-type embryos stained for MTs, DNA (left, green and red), and AIR-1 (right). A recently fertilized embryo (top) and a metaphase embryo (bottom) are shown. (C) A single deconvolved focal plane showing a centrosome from a metaphase embryo stained for γ-tubulin and AIR-1. (D) A mitotic air-1(RNAi) embryo stained for MTs, DNA (left, green and red), and AIR-1 (right). Bars: (B and D) 10 μm; (C) 2.5 μm.
Mentions: The C. elegans homologue of aurora-A, AIR-1, localizes to centrosomes and is required for normal spindle assembly (Schumacher et al., 1998). To investigate the role of aurora-A in the centrosome cycle, we first analyzed its localization during the first embryonic division. We raised an antibody to AIR-1 that detects a single band of ∼40 kD on Western blots. This band is reduced >90% in air-1(RNAi) worms (Fig. 1 A), confirming the specificity of our antibody. During fertilization, a sperm-derived centrosome enters the egg (which lacks centrosomes) and duplicates, resulting in two small centrosomes positioned between the sperm pronucleus and the cortex. AIR-1 localizes to centrosomes in early embryos (Fig. 1 B, top) and also weakly to astral and cytoplasmic MTs. In wild-type, the maternal pronucleus migrates toward the sperm pronucleus as the chromosomes condense. The pronuclei fuse, their nuclear envelopes break down, and the first mitotic spindle assembles. In mitotic embryos, AIR-1 concentrates in the centers of the asters (Fig. 1 B, bottom) in a donut-shaped region peripheral to γ-tubulin staining (Fig. 1 C) and also localizes along the base of astral MTs. The pattern of localization of AIR-1 is specific, since it is not observed in air-1(RNAi) embryos.

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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