Limits...
Interaction of Aurora-A and centrosomin at the microtubule-nucleating site in Drosophila and mammalian cells.

Terada Y, Uetake Y, Kuriyama R - J. Cell Biol. (2003)

Bottom Line: Aurora-A and CNN are mutually dependent for localization at spindle poles, which is required for proper targeting of gamma-tubulin and other centrosomal components to the centrosome.The NH2-terminal half of CNN interacts with gamma-tubulin, and induces cytoplasmic foci that can initiate microtubule nucleation in vivo and in vitro in both Drosophila and mammalian cells.These results suggest that Aurora-A regulates centrosome assembly by controlling the CNN's ability to targeting and/or anchoring gamma-tubulin to the centrosome and organizing microtubule-nucleating sites via its interaction with the COOH-terminal sequence of CNN.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN 55455, USA. terad002@umn.edu

ABSTRACT
A mitosis-specific Aurora-A kinase has been implicated in microtubule organization and spindle assembly in diverse organisms. However, exactly how Aurora-A controls the microtubule nucleation onto centrosomes is unknown. Here, we show that Aurora-A specifically binds to the COOH-terminal domain of a Drosophila centrosomal protein, centrosomin (CNN), which has been shown to be important for assembly of mitotic spindles and spindle poles. Aurora-A and CNN are mutually dependent for localization at spindle poles, which is required for proper targeting of gamma-tubulin and other centrosomal components to the centrosome. The NH2-terminal half of CNN interacts with gamma-tubulin, and induces cytoplasmic foci that can initiate microtubule nucleation in vivo and in vitro in both Drosophila and mammalian cells. These results suggest that Aurora-A regulates centrosome assembly by controlling the CNN's ability to targeting and/or anchoring gamma-tubulin to the centrosome and organizing microtubule-nucleating sites via its interaction with the COOH-terminal sequence of CNN.

Show MeSH

Related in: MedlinePlus

CNN interacts with γ-tubulin and induces microtubule-nucleating sites. (A) The NH2-terminal domain of CNN binds to γ-tubulin. Nickel beads conjugated with His-tagged full (F), NH2-terminal (N), and COOH-terminal (C) domains of CNN were incubated with cell extracts prepared from mitotic S2 (lanes 2–4) and CHO cells (lanes 6–8). Endogenous γ-tubulin is shown in lanes 1 and 5. (B–D) Immunostaining of HA-CNN–expressing S2 (B) and CHO cells (C–D) with HA (B–D) and α-tubulin antibodies (B′–D′). Merged images were shown in B′′–D′′. CNN expression caused the formation of cytoplasmic aggregates associated with microtubule asters. In D–D′′, cells were briefly recovered from nocodazole treatment before fixation. (E–G) Colocalization of pericentrin (E), Cep135 (F), and γ-tubulin (G) at the cytoplasmic foci induced by CNN expression. Merged images of CNN (green) and other centrosomal proteins (red) were shown. (H–I) Induction of GFP-tagged γ-tubulin in CHO cells coexpressing (H) or not coexpressing HA-CNN (I). To merge images, GFP was converted to red in double-stained cells with microtubules (green) and HA (green). Although expression of γ-tubulin alone induced cytoplasmic dots, microtubule-nucleating activity of the aggregates was detected only when γ-tubulin was coexpressed with CNN. Bars, 10 μm (B) and 50 μm (C–I).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172831&req=5

fig3: CNN interacts with γ-tubulin and induces microtubule-nucleating sites. (A) The NH2-terminal domain of CNN binds to γ-tubulin. Nickel beads conjugated with His-tagged full (F), NH2-terminal (N), and COOH-terminal (C) domains of CNN were incubated with cell extracts prepared from mitotic S2 (lanes 2–4) and CHO cells (lanes 6–8). Endogenous γ-tubulin is shown in lanes 1 and 5. (B–D) Immunostaining of HA-CNN–expressing S2 (B) and CHO cells (C–D) with HA (B–D) and α-tubulin antibodies (B′–D′). Merged images were shown in B′′–D′′. CNN expression caused the formation of cytoplasmic aggregates associated with microtubule asters. In D–D′′, cells were briefly recovered from nocodazole treatment before fixation. (E–G) Colocalization of pericentrin (E), Cep135 (F), and γ-tubulin (G) at the cytoplasmic foci induced by CNN expression. Merged images of CNN (green) and other centrosomal proteins (red) were shown. (H–I) Induction of GFP-tagged γ-tubulin in CHO cells coexpressing (H) or not coexpressing HA-CNN (I). To merge images, GFP was converted to red in double-stained cells with microtubules (green) and HA (green). Although expression of γ-tubulin alone induced cytoplasmic dots, microtubule-nucleating activity of the aggregates was detected only when γ-tubulin was coexpressed with CNN. Bars, 10 μm (B) and 50 μm (C–I).

Mentions: To confirm the role of CNN in recruiting γ-tubulin, we analyzed protein interaction in vitro. In Fig. 3 A (lanes 1–4), nickel beads conjugated with His-tagged CNN were mixed with cell extracts prepared from colcemid-treated S2 cells. γ-Tubulin was specifically sedimented by the full (Fig. 3 A, lane 2) and NH2-terminal (Fig. 3 A, lane 3) sequence, but not the COOH-terminal sequence (Fig. 3 A, lane 4) of CNN. Because neither in vitro binding assays nor two-hybrid screens demonstrated direct binding between two molecules (unpublished data), CNN may interact with a γ-tubulin complex, rather than γ-tubulin directly (Barbosa et al., 2000). Further, we expressed HA-tagged CNN in S2 cells. As shown in Fig. 3 (B–B′′), exogenous proteins caused formation of γ-tubulin–containing cytoplasmic aggregates capable of microtubule formation and association with microtubule asters. These results clearly indicate that the NH2-terminal domain of CNN interacts with γ-tubulin/γ-TuRC and plays an important role in assembly of MTOCs.


Interaction of Aurora-A and centrosomin at the microtubule-nucleating site in Drosophila and mammalian cells.

Terada Y, Uetake Y, Kuriyama R - J. Cell Biol. (2003)

CNN interacts with γ-tubulin and induces microtubule-nucleating sites. (A) The NH2-terminal domain of CNN binds to γ-tubulin. Nickel beads conjugated with His-tagged full (F), NH2-terminal (N), and COOH-terminal (C) domains of CNN were incubated with cell extracts prepared from mitotic S2 (lanes 2–4) and CHO cells (lanes 6–8). Endogenous γ-tubulin is shown in lanes 1 and 5. (B–D) Immunostaining of HA-CNN–expressing S2 (B) and CHO cells (C–D) with HA (B–D) and α-tubulin antibodies (B′–D′). Merged images were shown in B′′–D′′. CNN expression caused the formation of cytoplasmic aggregates associated with microtubule asters. In D–D′′, cells were briefly recovered from nocodazole treatment before fixation. (E–G) Colocalization of pericentrin (E), Cep135 (F), and γ-tubulin (G) at the cytoplasmic foci induced by CNN expression. Merged images of CNN (green) and other centrosomal proteins (red) were shown. (H–I) Induction of GFP-tagged γ-tubulin in CHO cells coexpressing (H) or not coexpressing HA-CNN (I). To merge images, GFP was converted to red in double-stained cells with microtubules (green) and HA (green). Although expression of γ-tubulin alone induced cytoplasmic dots, microtubule-nucleating activity of the aggregates was detected only when γ-tubulin was coexpressed with CNN. Bars, 10 μm (B) and 50 μm (C–I).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172831&req=5

fig3: CNN interacts with γ-tubulin and induces microtubule-nucleating sites. (A) The NH2-terminal domain of CNN binds to γ-tubulin. Nickel beads conjugated with His-tagged full (F), NH2-terminal (N), and COOH-terminal (C) domains of CNN were incubated with cell extracts prepared from mitotic S2 (lanes 2–4) and CHO cells (lanes 6–8). Endogenous γ-tubulin is shown in lanes 1 and 5. (B–D) Immunostaining of HA-CNN–expressing S2 (B) and CHO cells (C–D) with HA (B–D) and α-tubulin antibodies (B′–D′). Merged images were shown in B′′–D′′. CNN expression caused the formation of cytoplasmic aggregates associated with microtubule asters. In D–D′′, cells were briefly recovered from nocodazole treatment before fixation. (E–G) Colocalization of pericentrin (E), Cep135 (F), and γ-tubulin (G) at the cytoplasmic foci induced by CNN expression. Merged images of CNN (green) and other centrosomal proteins (red) were shown. (H–I) Induction of GFP-tagged γ-tubulin in CHO cells coexpressing (H) or not coexpressing HA-CNN (I). To merge images, GFP was converted to red in double-stained cells with microtubules (green) and HA (green). Although expression of γ-tubulin alone induced cytoplasmic dots, microtubule-nucleating activity of the aggregates was detected only when γ-tubulin was coexpressed with CNN. Bars, 10 μm (B) and 50 μm (C–I).
Mentions: To confirm the role of CNN in recruiting γ-tubulin, we analyzed protein interaction in vitro. In Fig. 3 A (lanes 1–4), nickel beads conjugated with His-tagged CNN were mixed with cell extracts prepared from colcemid-treated S2 cells. γ-Tubulin was specifically sedimented by the full (Fig. 3 A, lane 2) and NH2-terminal (Fig. 3 A, lane 3) sequence, but not the COOH-terminal sequence (Fig. 3 A, lane 4) of CNN. Because neither in vitro binding assays nor two-hybrid screens demonstrated direct binding between two molecules (unpublished data), CNN may interact with a γ-tubulin complex, rather than γ-tubulin directly (Barbosa et al., 2000). Further, we expressed HA-tagged CNN in S2 cells. As shown in Fig. 3 (B–B′′), exogenous proteins caused formation of γ-tubulin–containing cytoplasmic aggregates capable of microtubule formation and association with microtubule asters. These results clearly indicate that the NH2-terminal domain of CNN interacts with γ-tubulin/γ-TuRC and plays an important role in assembly of MTOCs.

Bottom Line: Aurora-A and CNN are mutually dependent for localization at spindle poles, which is required for proper targeting of gamma-tubulin and other centrosomal components to the centrosome.The NH2-terminal half of CNN interacts with gamma-tubulin, and induces cytoplasmic foci that can initiate microtubule nucleation in vivo and in vitro in both Drosophila and mammalian cells.These results suggest that Aurora-A regulates centrosome assembly by controlling the CNN's ability to targeting and/or anchoring gamma-tubulin to the centrosome and organizing microtubule-nucleating sites via its interaction with the COOH-terminal sequence of CNN.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN 55455, USA. terad002@umn.edu

ABSTRACT
A mitosis-specific Aurora-A kinase has been implicated in microtubule organization and spindle assembly in diverse organisms. However, exactly how Aurora-A controls the microtubule nucleation onto centrosomes is unknown. Here, we show that Aurora-A specifically binds to the COOH-terminal domain of a Drosophila centrosomal protein, centrosomin (CNN), which has been shown to be important for assembly of mitotic spindles and spindle poles. Aurora-A and CNN are mutually dependent for localization at spindle poles, which is required for proper targeting of gamma-tubulin and other centrosomal components to the centrosome. The NH2-terminal half of CNN interacts with gamma-tubulin, and induces cytoplasmic foci that can initiate microtubule nucleation in vivo and in vitro in both Drosophila and mammalian cells. These results suggest that Aurora-A regulates centrosome assembly by controlling the CNN's ability to targeting and/or anchoring gamma-tubulin to the centrosome and organizing microtubule-nucleating sites via its interaction with the COOH-terminal sequence of CNN.

Show MeSH
Related in: MedlinePlus