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

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.

Figure 1.

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.

Figure 2.

AIR-1 is required to maintain centrosome separation during spindle assembly. (A) Panels summarize time-lapse recordings of wild-type (left) and air-1(RNAi) embryos (right) expressing GFP–α-tubulin. Times are seconds after NEBD. In wild-type (left), unseparated asters (−446 s, arrow) associate with the sperm pronucleus. (−174 s) The asters separate and position themselves on opposite sides of the sperm pronucleus. The maternal pronucleus is also indicated (arrowhead). (+0 s) The pronuclei meet, move with the asters (arrows) to the center of the embryo, and the nuclear envelope breaks down. (+145) After NEBD, the asters increase in size and become the poles of the mitotic spindle. In air-1(RNAi) embryos (right), the asters (arrows) separate as in wild-type (−563 s and −282 s). The maternal pronucleus (arrowhead) is out of focus. After NEBD, the centrosomal asters (arrows) collapse toward each other till they sit side by side (+97 s and +177 s). See also videos 1 and 2 available at http://www.jcb.org/cgi/ content/full/jcb.200108051/DC1. (B) Kinetic traces of centrosome separation. Centrosome separation was measured for each time point where both centrosomes were in focus in seven wild-type and seven air-1(RNAi) embryos. Three examples are shown for each. Times are with respect to NEBD. (C) Kinetic traces of centrosomal α-tubulin fluorescence. Centrosomal fluorescence was quantitated in five wild-type and six air-1(RNAi) embryos. Three traces are shown for each. Bar, 10 μm.

Figure 3.

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).

Figure 4.

AIR-1 is required for the accumulation of γ-tubulin, CeGrip, and ZYG-9 during centrosome maturation. Wild-type and air-1(RNAi) embryos stained for MTs and DNA (left, green and red) and for either γ-tubulin (A), CeGrip (B), or ZYG-9 (C) are shown. Inset in A is magnified 5.5-fold. Bar, 10 μm.

Figure 5.

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.