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
Cytoplasmic aggregates induced by CNN overexpression in mammalian cells. (A–D) In vitro microtubule nucleation onto CNN-containing sites detected by phase-contrast (A) and fluorescence (B–D) microscopy. GFP-tagged CNN aggregates were fractionated from CHO cells and incubated with X-rhodamine–conjugated brain tubulin. CNN aggregates in different sizes and shapes nucleated various numbers of microtubules. (E) Time-lapse images of GFP-tagged CNN-containing aggregates with assembled microtubules. CNN aggregates mixed with X-rhodamine tubulin were placed on a microscopic stage at time zero, and fluorescence images were taken at indicated times after the temperature was shifted to 37°C. (F) Thin-section EM of CHO cells expressing GFP-tagged CNN. The cells were briefly extracted with a detergent containing microtubule-stabilizing buffer before fixation. Two microtubule asters are seen in the field, and there is an electron-dense particle of different shape at each center. F′ and F′′ are close-ups of the areas outlined in F. (G) Immunofluorescence staining of 293 cells with anti-human centrin-2 antibodies (red). Dotted lines indicate the outline of a cell expressing CNN aggregates (green). There are two centrioles (arrows) that were not included in all sites induced by CNN overexpression. Bars, 10 μm (A–E, and G) and 1 μm (F–F′′).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172831&req=5

fig4: Cytoplasmic aggregates induced by CNN overexpression in mammalian cells. (A–D) In vitro microtubule nucleation onto CNN-containing sites detected by phase-contrast (A) and fluorescence (B–D) microscopy. GFP-tagged CNN aggregates were fractionated from CHO cells and incubated with X-rhodamine–conjugated brain tubulin. CNN aggregates in different sizes and shapes nucleated various numbers of microtubules. (E) Time-lapse images of GFP-tagged CNN-containing aggregates with assembled microtubules. CNN aggregates mixed with X-rhodamine tubulin were placed on a microscopic stage at time zero, and fluorescence images were taken at indicated times after the temperature was shifted to 37°C. (F) Thin-section EM of CHO cells expressing GFP-tagged CNN. The cells were briefly extracted with a detergent containing microtubule-stabilizing buffer before fixation. Two microtubule asters are seen in the field, and there is an electron-dense particle of different shape at each center. F′ and F′′ are close-ups of the areas outlined in F. (G) Immunofluorescence staining of 293 cells with anti-human centrin-2 antibodies (red). Dotted lines indicate the outline of a cell expressing CNN aggregates (green). There are two centrioles (arrows) that were not included in all sites induced by CNN overexpression. Bars, 10 μm (A–E, and G) and 1 μm (F–F′′).

Mentions: To confirm the microtubule-nucleating activity of the CNN aggregates, we polymerized microtubules in vitro by incubating isolated GFP-tagged CNN dots with X-rhodamine–conjugated brain tubulin. Fig. 4 (A–D) shows microtubule asters detected by phase-contrast and fluorescence microscopy. There is always a dot positive in GFP fluorescence at the center of the microtubule asters. Although variable numbers of microtubules emanated from the center, more microtubules tended to polymerize onto the GFP dots in larger sizes (Fig. 4, B–D). In Fig. 4 E, the process of aster formation was monitored by time-lapse microscopy. A fluorescence image taken 10 min after mounting the sample on a microscopic stage revealed several microtubules growing from a GFP-positive site. As time progressed, more microtubules appeared to emanate from the center, indicating that microtubules were formed by direct polymerization onto the CNN-containing foci, rather than that preformed microtubules were gathered around the center.


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)

Cytoplasmic aggregates induced by CNN overexpression in mammalian cells. (A–D) In vitro microtubule nucleation onto CNN-containing sites detected by phase-contrast (A) and fluorescence (B–D) microscopy. GFP-tagged CNN aggregates were fractionated from CHO cells and incubated with X-rhodamine–conjugated brain tubulin. CNN aggregates in different sizes and shapes nucleated various numbers of microtubules. (E) Time-lapse images of GFP-tagged CNN-containing aggregates with assembled microtubules. CNN aggregates mixed with X-rhodamine tubulin were placed on a microscopic stage at time zero, and fluorescence images were taken at indicated times after the temperature was shifted to 37°C. (F) Thin-section EM of CHO cells expressing GFP-tagged CNN. The cells were briefly extracted with a detergent containing microtubule-stabilizing buffer before fixation. Two microtubule asters are seen in the field, and there is an electron-dense particle of different shape at each center. F′ and F′′ are close-ups of the areas outlined in F. (G) Immunofluorescence staining of 293 cells with anti-human centrin-2 antibodies (red). Dotted lines indicate the outline of a cell expressing CNN aggregates (green). There are two centrioles (arrows) that were not included in all sites induced by CNN overexpression. Bars, 10 μm (A–E, and G) and 1 μm (F–F′′).
© Copyright Policy
Related In: Results  -  Collection

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

fig4: Cytoplasmic aggregates induced by CNN overexpression in mammalian cells. (A–D) In vitro microtubule nucleation onto CNN-containing sites detected by phase-contrast (A) and fluorescence (B–D) microscopy. GFP-tagged CNN aggregates were fractionated from CHO cells and incubated with X-rhodamine–conjugated brain tubulin. CNN aggregates in different sizes and shapes nucleated various numbers of microtubules. (E) Time-lapse images of GFP-tagged CNN-containing aggregates with assembled microtubules. CNN aggregates mixed with X-rhodamine tubulin were placed on a microscopic stage at time zero, and fluorescence images were taken at indicated times after the temperature was shifted to 37°C. (F) Thin-section EM of CHO cells expressing GFP-tagged CNN. The cells were briefly extracted with a detergent containing microtubule-stabilizing buffer before fixation. Two microtubule asters are seen in the field, and there is an electron-dense particle of different shape at each center. F′ and F′′ are close-ups of the areas outlined in F. (G) Immunofluorescence staining of 293 cells with anti-human centrin-2 antibodies (red). Dotted lines indicate the outline of a cell expressing CNN aggregates (green). There are two centrioles (arrows) that were not included in all sites induced by CNN overexpression. Bars, 10 μm (A–E, and G) and 1 μm (F–F′′).
Mentions: To confirm the microtubule-nucleating activity of the CNN aggregates, we polymerized microtubules in vitro by incubating isolated GFP-tagged CNN dots with X-rhodamine–conjugated brain tubulin. Fig. 4 (A–D) shows microtubule asters detected by phase-contrast and fluorescence microscopy. There is always a dot positive in GFP fluorescence at the center of the microtubule asters. Although variable numbers of microtubules emanated from the center, more microtubules tended to polymerize onto the GFP dots in larger sizes (Fig. 4, B–D). In Fig. 4 E, the process of aster formation was monitored by time-lapse microscopy. A fluorescence image taken 10 min after mounting the sample on a microscopic stage revealed several microtubules growing from a GFP-positive site. As time progressed, more microtubules appeared to emanate from the center, indicating that microtubules were formed by direct polymerization onto the CNN-containing foci, rather than that preformed microtubules were gathered around the center.

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