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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 is required for the accumulation of centrosomal γ-tubulin during maturation. (A) Panels summarize time-lapse recordings of wild-type (left) and air-1(RNAi) embryos (right) expressing GFP histone and GFP–γ-tubulin. Both sequences start at a similar early embryonic stage, judged by the extent of cortical ruffling, the small size of the sperm pronuclei (arrowheads), and the lack of separation of γ-tubulin–labeled centrosomes (arrow). The sequences end with onset of cytokinesis (wild-type, +242 s) or cortical contractions (air-1(RNAi), +121 s, arrowheads). The duration of both recordings is ∼12 min. Times are seconds after NEBD. The times in the corresponding panels differ by ∼2 min because NEBD is slightly delayed in air-1(RNAi) embryos. See videos 3–6 available at http://www.jcb.org/cgi/content/full/jcb.200108051/DC1. (Left) In wild-type, the DNA condenses, beginning before the migration of the pronuclei toward each other and continuing during migration. This occurs concomitant with an increase in the amount of centrosomal γ-tubulin (compare −256 s with −88 s). The accumulation of centrosomal γ-tubulin continues after NEBD as the mitotic spindle assembles (+122 s). (Right) In air-1(RNAi) embryos, the chromosomes condense with timing similar to wild-type, but γ-tubulin fails to accumulate at centrosomes (arrows, all panels). The migration of the maternal pronucleus toward the sperm pronucleus is lethargic, probably due to the failure of centrosomes to nucleate robust mitotic asters, but the two pronuclei eventually move toward each other, and the nuclear envelope breaks down (0 s). Chromosomes never align properly, but cortical contractions begin coincident with cytokinesis in wild-type embryos (+121 s). Cytokinesis does not succeed in air-1(RNAi) embryos (unpublished data). (B) Kinetic traces of centrosomal γ-tubulin fluorescence. Centrosomal fluorescence was quantified in 7 wild-type and 10 air-1(RNAi) embryos. Three traces are shown for each. Bar, 10 μm (as in Fig. 2).
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fig3: AIR-1 is required for the accumulation of centrosomal γ-tubulin during maturation. (A) Panels summarize time-lapse recordings of wild-type (left) and air-1(RNAi) embryos (right) expressing GFP histone and GFP–γ-tubulin. Both sequences start at a similar early embryonic stage, judged by the extent of cortical ruffling, the small size of the sperm pronuclei (arrowheads), and the lack of separation of γ-tubulin–labeled centrosomes (arrow). The sequences end with onset of cytokinesis (wild-type, +242 s) or cortical contractions (air-1(RNAi), +121 s, arrowheads). The duration of both recordings is ∼12 min. Times are seconds after NEBD. The times in the corresponding panels differ by ∼2 min because NEBD is slightly delayed in air-1(RNAi) embryos. See videos 3–6 available at http://www.jcb.org/cgi/content/full/jcb.200108051/DC1. (Left) In wild-type, the DNA condenses, beginning before the migration of the pronuclei toward each other and continuing during migration. This occurs concomitant with an increase in the amount of centrosomal γ-tubulin (compare −256 s with −88 s). The accumulation of centrosomal γ-tubulin continues after NEBD as the mitotic spindle assembles (+122 s). (Right) In air-1(RNAi) embryos, the chromosomes condense with timing similar to wild-type, but γ-tubulin fails to accumulate at centrosomes (arrows, all panels). The migration of the maternal pronucleus toward the sperm pronucleus is lethargic, probably due to the failure of centrosomes to nucleate robust mitotic asters, but the two pronuclei eventually move toward each other, and the nuclear envelope breaks down (0 s). Chromosomes never align properly, but cortical contractions begin coincident with cytokinesis in wild-type embryos (+121 s). Cytokinesis does not succeed in air-1(RNAi) embryos (unpublished data). (B) Kinetic traces of centrosomal γ-tubulin fluorescence. Centrosomal fluorescence was quantified in 7 wild-type and 10 air-1(RNAi) embryos. Three traces are shown for each. Bar, 10 μm (as in Fig. 2).

Mentions: The small centrosomal asters in mitotic air-1(RNAi) embryos suggested a role for AIR-1 in centrosome maturation. To determine if AIR-1 has a role in the accumulation of centrosomal γ-tubulin during this transition, we filmed air-1(RNAi) embryos expressing both GFP histone, as a marker for cell cycle progression, and GFP–γ-tubulin (Fig. 3 A). γ-Tubulin was visible shortly after fertilization at the still tiny centrosomes in both wild-type and air-1(RNAi) embryos (Fig. 3 A, −484 s and −608 s). The cell cycle progressed in air-1(RNAi) embryos as evidenced by DNA condensation, the release of cortical contraction at the end of pseudocleavage, NEBD, the onset of cortical contractions coincident with the initiation of cytokinesis in wild-type, and reformation of the nuclear envelope. However, the dramatic accumulation of centrosomal γ-tubulin that occurs in wild-type during the interval between 300 s before NEBD and anaphase onset (Fig. 3 A, compare –256 s with +122 s) was completely absent in air-1(RNAi) embryos (Fig. 3 A, right, all panels). Quantification of integrated fluorescence intensity showed that centrosomal γ-tubulin fluorescence does not change significantly as air-1(RNAi) embryos enter mitosis (Fig. 3 B). In summary, our results show that the cell cycle progresses in air-1(RNAi) embryos. However, the dramatic increase in centrosomal γ-tubulin, a hallmark of centrosome maturation, never occurs.


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 is required for the accumulation of centrosomal γ-tubulin during maturation. (A) Panels summarize time-lapse recordings of wild-type (left) and air-1(RNAi) embryos (right) expressing GFP histone and GFP–γ-tubulin. Both sequences start at a similar early embryonic stage, judged by the extent of cortical ruffling, the small size of the sperm pronuclei (arrowheads), and the lack of separation of γ-tubulin–labeled centrosomes (arrow). The sequences end with onset of cytokinesis (wild-type, +242 s) or cortical contractions (air-1(RNAi), +121 s, arrowheads). The duration of both recordings is ∼12 min. Times are seconds after NEBD. The times in the corresponding panels differ by ∼2 min because NEBD is slightly delayed in air-1(RNAi) embryos. See videos 3–6 available at http://www.jcb.org/cgi/content/full/jcb.200108051/DC1. (Left) In wild-type, the DNA condenses, beginning before the migration of the pronuclei toward each other and continuing during migration. This occurs concomitant with an increase in the amount of centrosomal γ-tubulin (compare −256 s with −88 s). The accumulation of centrosomal γ-tubulin continues after NEBD as the mitotic spindle assembles (+122 s). (Right) In air-1(RNAi) embryos, the chromosomes condense with timing similar to wild-type, but γ-tubulin fails to accumulate at centrosomes (arrows, all panels). The migration of the maternal pronucleus toward the sperm pronucleus is lethargic, probably due to the failure of centrosomes to nucleate robust mitotic asters, but the two pronuclei eventually move toward each other, and the nuclear envelope breaks down (0 s). Chromosomes never align properly, but cortical contractions begin coincident with cytokinesis in wild-type embryos (+121 s). Cytokinesis does not succeed in air-1(RNAi) embryos (unpublished data). (B) Kinetic traces of centrosomal γ-tubulin fluorescence. Centrosomal fluorescence was quantified in 7 wild-type and 10 air-1(RNAi) embryos. Three traces are shown for each. Bar, 10 μm (as in Fig. 2).
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fig3: AIR-1 is required for the accumulation of centrosomal γ-tubulin during maturation. (A) Panels summarize time-lapse recordings of wild-type (left) and air-1(RNAi) embryos (right) expressing GFP histone and GFP–γ-tubulin. Both sequences start at a similar early embryonic stage, judged by the extent of cortical ruffling, the small size of the sperm pronuclei (arrowheads), and the lack of separation of γ-tubulin–labeled centrosomes (arrow). The sequences end with onset of cytokinesis (wild-type, +242 s) or cortical contractions (air-1(RNAi), +121 s, arrowheads). The duration of both recordings is ∼12 min. Times are seconds after NEBD. The times in the corresponding panels differ by ∼2 min because NEBD is slightly delayed in air-1(RNAi) embryos. See videos 3–6 available at http://www.jcb.org/cgi/content/full/jcb.200108051/DC1. (Left) In wild-type, the DNA condenses, beginning before the migration of the pronuclei toward each other and continuing during migration. This occurs concomitant with an increase in the amount of centrosomal γ-tubulin (compare −256 s with −88 s). The accumulation of centrosomal γ-tubulin continues after NEBD as the mitotic spindle assembles (+122 s). (Right) In air-1(RNAi) embryos, the chromosomes condense with timing similar to wild-type, but γ-tubulin fails to accumulate at centrosomes (arrows, all panels). The migration of the maternal pronucleus toward the sperm pronucleus is lethargic, probably due to the failure of centrosomes to nucleate robust mitotic asters, but the two pronuclei eventually move toward each other, and the nuclear envelope breaks down (0 s). Chromosomes never align properly, but cortical contractions begin coincident with cytokinesis in wild-type embryos (+121 s). Cytokinesis does not succeed in air-1(RNAi) embryos (unpublished data). (B) Kinetic traces of centrosomal γ-tubulin fluorescence. Centrosomal fluorescence was quantified in 7 wild-type and 10 air-1(RNAi) embryos. Three traces are shown for each. Bar, 10 μm (as in Fig. 2).
Mentions: The small centrosomal asters in mitotic air-1(RNAi) embryos suggested a role for AIR-1 in centrosome maturation. To determine if AIR-1 has a role in the accumulation of centrosomal γ-tubulin during this transition, we filmed air-1(RNAi) embryos expressing both GFP histone, as a marker for cell cycle progression, and GFP–γ-tubulin (Fig. 3 A). γ-Tubulin was visible shortly after fertilization at the still tiny centrosomes in both wild-type and air-1(RNAi) embryos (Fig. 3 A, −484 s and −608 s). The cell cycle progressed in air-1(RNAi) embryos as evidenced by DNA condensation, the release of cortical contraction at the end of pseudocleavage, NEBD, the onset of cortical contractions coincident with the initiation of cytokinesis in wild-type, and reformation of the nuclear envelope. However, the dramatic accumulation of centrosomal γ-tubulin that occurs in wild-type during the interval between 300 s before NEBD and anaphase onset (Fig. 3 A, compare –256 s with +122 s) was completely absent in air-1(RNAi) embryos (Fig. 3 A, right, all panels). Quantification of integrated fluorescence intensity showed that centrosomal γ-tubulin fluorescence does not change significantly as air-1(RNAi) embryos enter mitosis (Fig. 3 B). In summary, our results show that the cell cycle progresses in air-1(RNAi) embryos. However, the dramatic increase in centrosomal γ-tubulin, a hallmark of centrosome maturation, never occurs.

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
Related in: MedlinePlus