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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.

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Immunolocalization of centrosome proteins at the spindle pole. (A) Double staining of mitotic S2 cells with Aurora-A/γ-tubulin and CNN/γ-tubulin after depletion of either Aurora-A (a–f) or CNN (g–l) by RNAi. DAPI staining is also shown in cells labeled with γ-tubulin (gamma). Different amounts of Aurora-A and CNN remained at the pole in depleted cells. Aurora-A and CNN are dependent on one another to localize at the spindle pole, and two proteins are required for recruiting γ-tubulin to the centrosome. (B) Localization of CP190 (a′–c′) and CP60 (d′–f′) in control (a and d), Aurora-A–depleted (b and e), and CNN-depleted (c and f) cells. The cells were also stained with α-tubulin and DAPI (a–f). Although depletion of Aurora-A and CNN predominantly induced abnormal spindles in monopolar/multipolar organization, spindles in bipolar orientation were selected to demonstrate the absence of centrosomal proteins at each pole. Bars, 10 μm.
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fig2: Immunolocalization of centrosome proteins at the spindle pole. (A) Double staining of mitotic S2 cells with Aurora-A/γ-tubulin and CNN/γ-tubulin after depletion of either Aurora-A (a–f) or CNN (g–l) by RNAi. DAPI staining is also shown in cells labeled with γ-tubulin (gamma). Different amounts of Aurora-A and CNN remained at the pole in depleted cells. Aurora-A and CNN are dependent on one another to localize at the spindle pole, and two proteins are required for recruiting γ-tubulin to the centrosome. (B) Localization of CP190 (a′–c′) and CP60 (d′–f′) in control (a and d), Aurora-A–depleted (b and e), and CNN-depleted (c and f) cells. The cells were also stained with α-tubulin and DAPI (a–f). Although depletion of Aurora-A and CNN predominantly induced abnormal spindles in monopolar/multipolar organization, spindles in bipolar orientation were selected to demonstrate the absence of centrosomal proteins at each pole. Bars, 10 μm.

Mentions: To investigate the role of protein interaction in the centrosome, we prepared S2 cells from which Aurora-A or CNN was depleted by RNA interference (RNAi; Fig. 2 A). In cells lacking Aurora-A (a), not only CNN (d′), but also γ-tubulin (a′ and d), were absent at each spindle pole, which agrees with a previous report (Berdnik and Knoblich, 2002). When CNN was depleted (g), neither γ-tubulin (g′ and j; Megraw et al., 1999; Vaizel-Ohayon and Schejter, 1999) nor Aurora-A (j′) was seen at the spindle pole. In cells with partially depleted Aurora-A (b) or CNN (h), we detected comparable amounts of γ-tubulin (b′, e, h′, and k) and CNN (e′) or Aurora-A (k′) at each pole. Fig. 2 B demonstrates that, besides γ-tubulin, other centrosome proteins, CP190 (a–c) and CP60 (d–f), became dislocated from the spindle poles in RNAi cells. Therefore, we concluded that CNN and Aurora-A are mutually dependent for localization at spindle poles, which is required for proper targeting of other centrosomal proteins to the centrosome. This is consistent with previous observations that the centrosomal association of CNN is not dependent on the presence of γ-tubulin/γ-TuRC (Barbosa et al., 2000).


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)

Immunolocalization of centrosome proteins at the spindle pole. (A) Double staining of mitotic S2 cells with Aurora-A/γ-tubulin and CNN/γ-tubulin after depletion of either Aurora-A (a–f) or CNN (g–l) by RNAi. DAPI staining is also shown in cells labeled with γ-tubulin (gamma). Different amounts of Aurora-A and CNN remained at the pole in depleted cells. Aurora-A and CNN are dependent on one another to localize at the spindle pole, and two proteins are required for recruiting γ-tubulin to the centrosome. (B) Localization of CP190 (a′–c′) and CP60 (d′–f′) in control (a and d), Aurora-A–depleted (b and e), and CNN-depleted (c and f) cells. The cells were also stained with α-tubulin and DAPI (a–f). Although depletion of Aurora-A and CNN predominantly induced abnormal spindles in monopolar/multipolar organization, spindles in bipolar orientation were selected to demonstrate the absence of centrosomal proteins at each pole. Bars, 10 μm.
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Related In: Results  -  Collection

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fig2: Immunolocalization of centrosome proteins at the spindle pole. (A) Double staining of mitotic S2 cells with Aurora-A/γ-tubulin and CNN/γ-tubulin after depletion of either Aurora-A (a–f) or CNN (g–l) by RNAi. DAPI staining is also shown in cells labeled with γ-tubulin (gamma). Different amounts of Aurora-A and CNN remained at the pole in depleted cells. Aurora-A and CNN are dependent on one another to localize at the spindle pole, and two proteins are required for recruiting γ-tubulin to the centrosome. (B) Localization of CP190 (a′–c′) and CP60 (d′–f′) in control (a and d), Aurora-A–depleted (b and e), and CNN-depleted (c and f) cells. The cells were also stained with α-tubulin and DAPI (a–f). Although depletion of Aurora-A and CNN predominantly induced abnormal spindles in monopolar/multipolar organization, spindles in bipolar orientation were selected to demonstrate the absence of centrosomal proteins at each pole. Bars, 10 μm.
Mentions: To investigate the role of protein interaction in the centrosome, we prepared S2 cells from which Aurora-A or CNN was depleted by RNA interference (RNAi; Fig. 2 A). In cells lacking Aurora-A (a), not only CNN (d′), but also γ-tubulin (a′ and d), were absent at each spindle pole, which agrees with a previous report (Berdnik and Knoblich, 2002). When CNN was depleted (g), neither γ-tubulin (g′ and j; Megraw et al., 1999; Vaizel-Ohayon and Schejter, 1999) nor Aurora-A (j′) was seen at the spindle pole. In cells with partially depleted Aurora-A (b) or CNN (h), we detected comparable amounts of γ-tubulin (b′, e, h′, and k) and CNN (e′) or Aurora-A (k′) at each pole. Fig. 2 B demonstrates that, besides γ-tubulin, other centrosome proteins, CP190 (a–c) and CP60 (d–f), became dislocated from the spindle poles in RNAi cells. Therefore, we concluded that CNN and Aurora-A are mutually dependent for localization at spindle poles, which is required for proper targeting of other centrosomal proteins to the centrosome. This is consistent with previous observations that the centrosomal association of CNN is not dependent on the presence of γ-tubulin/γ-TuRC (Barbosa et al., 2000).

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