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When overexpressed, a novel centrosomal protein, RanBPM, causes ectopic microtubule nucleation similar to gamma-tubulin.

Nakamura M, Masuda H, Horii J, Kuma Ki, Yokoyama N, Ohba T, Nishitani H, Miyata T, Tanaka M, Nishimoto T - J. Cell Biol. (1998)

Bottom Line: Furthermore, Saccharomyces cerevisiae was found to have a gene, YGL227w, the COOH-terminal half of which is 30% identical to RanBPM.Overexpression of RanBPM produced multiple spots which were colocalized with gamma-tubulin and acted as ectopic microtubule nucleation sites, resulting in a reorganization of microtubule network.These results provide evidence that the Ran-binding protein, RanBPM, is involved in microtubule nucleation, thereby suggesting that Ran regulates the centrosome through RanBPM.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Fukuoka 812-82, Japan.

ABSTRACT
A novel human protein with a molecular mass of 55 kD, designated RanBPM, was isolated with the two-hybrid method using Ran as a bait. Mouse and hamster RanBPM possessed a polypeptide identical to the human one. Furthermore, Saccharomyces cerevisiae was found to have a gene, YGL227w, the COOH-terminal half of which is 30% identical to RanBPM. Anti-RanBPM antibodies revealed that RanBPM was localized within the centrosome throughout the cell cycle. Overexpression of RanBPM produced multiple spots which were colocalized with gamma-tubulin and acted as ectopic microtubule nucleation sites, resulting in a reorganization of microtubule network. RanBPM cosedimented with the centrosomal fractions by sucrose- density gradient centrifugation. The formation of microtubule asters was inhibited not only by anti- RanBPM antibodies, but also by nonhydrolyzable GTP-Ran. Indeed, RanBPM specifically interacted with GTP-Ran in two-hybrid assay. The central part of asters stained by anti-RanBPM antibodies or by the mAb to gamma-tubulin was faded by the addition of GTPgammaS-Ran, but not by the addition of anti-RanBPM anti- bodies. These results provide evidence that the Ran-binding protein, RanBPM, is involved in microtubule nucleation, thereby suggesting that Ran regulates the centrosome through RanBPM.

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Ectopic microtubule nucleation in COS cells transfected with RanBPM cDNA. Cultures of COS cells growing on the coverslip  were transfected with 10 ng of pcDEB-T7–RanBPM per dishes (35-mm-diam). 24 h later, transfected cells were incubated on ice for 1 h  and then either processed immediately for immunofluorescence (0 time point of recovery), or else the cold medium was replaced with  warm (30°C) medium. Coverslips were harvested for immunofluorescence at 1, 2, or 5 min after medium change as indicated. Cells were  fixed and stained with the affinity-purified anti-RanBPM antibodies (red) and the mAb to α-tubulin (green). Both staining patterns  were superimposed by electronic image processing (superimposition). Bars, 10 μm.
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Figure 4: Ectopic microtubule nucleation in COS cells transfected with RanBPM cDNA. Cultures of COS cells growing on the coverslip were transfected with 10 ng of pcDEB-T7–RanBPM per dishes (35-mm-diam). 24 h later, transfected cells were incubated on ice for 1 h and then either processed immediately for immunofluorescence (0 time point of recovery), or else the cold medium was replaced with warm (30°C) medium. Coverslips were harvested for immunofluorescence at 1, 2, or 5 min after medium change as indicated. Cells were fixed and stained with the affinity-purified anti-RanBPM antibodies (red) and the mAb to α-tubulin (green). Both staining patterns were superimposed by electronic image processing (superimposition). Bars, 10 μm.

Mentions: To determine the biological function of RanBPM, we overexpressed RanBPM cDNA in COS cells and examined its effect on the microtubule network. T7-fused RanBPM cDNA carried on the pcDEB vector was introduced into COS cells as described in Materials and Methods. 36 h later, transfected cells were fixed and doubly stained by the affinity-purified anti-RanBPM antibodies (red) and by the mAb to α-tubulin (green). In contrast to cells transfected with the vector alone (Fig. 3 A, panel c), a normal radial network of microtubules was broken in cells transfected with T7-RanBPM cDNA (the representative figures are shown in Fig. 3 A, panels a and b). When transfected cells were doubly stained by the mAb to the T7-tag (green) and the affinity purified anti–γ-tubulin antibodies (red), RanBPM and γ-tubulin were found to be distributed as spots throughout the cytoplasm (Fig. 3 B). Both staining spots were colocalized when superimposed (Fig. 3 B, superimposition), indicating that there is some type of interaction between γ-tubulin and RanBPM. We thought that upon overexpression of T7-RanBPM, γ-tubulin was recruited onto RanBPM, resulting in reorganization of the microtubule network similar to the case of overexpressed γ-tubulin (Shu and Joshi, 1995). To confirm this issue, we monitored the recovery of microtubules after complete disassembly induced by lowering the temperature to 0°C as described (Joshi et al., 1992; Shu and Joshi, 1995). RanBPM cDNA or, as a control, the vector alone, was transfected into COS cells. To avoid rapid cell death, we transfected a smaller amount of RanBPM cDNA per cell (10 ng/dish), compared with the experiment described above (1 μg/dish). Under this condition, the average number of RanBPM-staining spots was ∼3–5 per cell. 24 h later, transfected cells were placed on ice for 1 h. Cells were then incubated in fresh medium at 30°C for varying time periods ranging from 0 to 5 min, lysed to remove free tubulin and then fixed to visualize the initiation of microtubule assembly sites in the green channel and RanBPM in the red channel by double immunofluorescence microscopy (Fig. 4). In cells transfected by the vector alone, after return to 30°C, short microtubules emerged from a single RanBPM-stained spot and became progressively elongated as previously reported (Shu and Joshi, 1995). In contrast to cells transfected by the vector alone, multiple RanBPM-stained spots appeared in cells transfected with T7-tagged RanBPM cDNA (Fig. 4). After incubation at 30°C for 1 min, α-tubulin gathered around multiple RanBPM-stained spots. Subsequently, short microtubules emerged from the multiple RanBPM-stained spots, although the microtubules were shorter than the microtubules that emerged from the centrosome of the untransfected cells. The number of ectopically nucleated microtubules was the same as the number of RanBPM-stained spots. Thus, we concluded that overexpressed T7-tagged RanBPM caused ectopic nucleation of the microtubule assembly in vivo.


When overexpressed, a novel centrosomal protein, RanBPM, causes ectopic microtubule nucleation similar to gamma-tubulin.

Nakamura M, Masuda H, Horii J, Kuma Ki, Yokoyama N, Ohba T, Nishitani H, Miyata T, Tanaka M, Nishimoto T - J. Cell Biol. (1998)

Ectopic microtubule nucleation in COS cells transfected with RanBPM cDNA. Cultures of COS cells growing on the coverslip  were transfected with 10 ng of pcDEB-T7–RanBPM per dishes (35-mm-diam). 24 h later, transfected cells were incubated on ice for 1 h  and then either processed immediately for immunofluorescence (0 time point of recovery), or else the cold medium was replaced with  warm (30°C) medium. Coverslips were harvested for immunofluorescence at 1, 2, or 5 min after medium change as indicated. Cells were  fixed and stained with the affinity-purified anti-RanBPM antibodies (red) and the mAb to α-tubulin (green). Both staining patterns  were superimposed by electronic image processing (superimposition). Bars, 10 μm.
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Related In: Results  -  Collection

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Figure 4: Ectopic microtubule nucleation in COS cells transfected with RanBPM cDNA. Cultures of COS cells growing on the coverslip were transfected with 10 ng of pcDEB-T7–RanBPM per dishes (35-mm-diam). 24 h later, transfected cells were incubated on ice for 1 h and then either processed immediately for immunofluorescence (0 time point of recovery), or else the cold medium was replaced with warm (30°C) medium. Coverslips were harvested for immunofluorescence at 1, 2, or 5 min after medium change as indicated. Cells were fixed and stained with the affinity-purified anti-RanBPM antibodies (red) and the mAb to α-tubulin (green). Both staining patterns were superimposed by electronic image processing (superimposition). Bars, 10 μm.
Mentions: To determine the biological function of RanBPM, we overexpressed RanBPM cDNA in COS cells and examined its effect on the microtubule network. T7-fused RanBPM cDNA carried on the pcDEB vector was introduced into COS cells as described in Materials and Methods. 36 h later, transfected cells were fixed and doubly stained by the affinity-purified anti-RanBPM antibodies (red) and by the mAb to α-tubulin (green). In contrast to cells transfected with the vector alone (Fig. 3 A, panel c), a normal radial network of microtubules was broken in cells transfected with T7-RanBPM cDNA (the representative figures are shown in Fig. 3 A, panels a and b). When transfected cells were doubly stained by the mAb to the T7-tag (green) and the affinity purified anti–γ-tubulin antibodies (red), RanBPM and γ-tubulin were found to be distributed as spots throughout the cytoplasm (Fig. 3 B). Both staining spots were colocalized when superimposed (Fig. 3 B, superimposition), indicating that there is some type of interaction between γ-tubulin and RanBPM. We thought that upon overexpression of T7-RanBPM, γ-tubulin was recruited onto RanBPM, resulting in reorganization of the microtubule network similar to the case of overexpressed γ-tubulin (Shu and Joshi, 1995). To confirm this issue, we monitored the recovery of microtubules after complete disassembly induced by lowering the temperature to 0°C as described (Joshi et al., 1992; Shu and Joshi, 1995). RanBPM cDNA or, as a control, the vector alone, was transfected into COS cells. To avoid rapid cell death, we transfected a smaller amount of RanBPM cDNA per cell (10 ng/dish), compared with the experiment described above (1 μg/dish). Under this condition, the average number of RanBPM-staining spots was ∼3–5 per cell. 24 h later, transfected cells were placed on ice for 1 h. Cells were then incubated in fresh medium at 30°C for varying time periods ranging from 0 to 5 min, lysed to remove free tubulin and then fixed to visualize the initiation of microtubule assembly sites in the green channel and RanBPM in the red channel by double immunofluorescence microscopy (Fig. 4). In cells transfected by the vector alone, after return to 30°C, short microtubules emerged from a single RanBPM-stained spot and became progressively elongated as previously reported (Shu and Joshi, 1995). In contrast to cells transfected by the vector alone, multiple RanBPM-stained spots appeared in cells transfected with T7-tagged RanBPM cDNA (Fig. 4). After incubation at 30°C for 1 min, α-tubulin gathered around multiple RanBPM-stained spots. Subsequently, short microtubules emerged from the multiple RanBPM-stained spots, although the microtubules were shorter than the microtubules that emerged from the centrosome of the untransfected cells. The number of ectopically nucleated microtubules was the same as the number of RanBPM-stained spots. Thus, we concluded that overexpressed T7-tagged RanBPM caused ectopic nucleation of the microtubule assembly in vivo.

Bottom Line: Furthermore, Saccharomyces cerevisiae was found to have a gene, YGL227w, the COOH-terminal half of which is 30% identical to RanBPM.Overexpression of RanBPM produced multiple spots which were colocalized with gamma-tubulin and acted as ectopic microtubule nucleation sites, resulting in a reorganization of microtubule network.These results provide evidence that the Ran-binding protein, RanBPM, is involved in microtubule nucleation, thereby suggesting that Ran regulates the centrosome through RanBPM.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Fukuoka 812-82, Japan.

ABSTRACT
A novel human protein with a molecular mass of 55 kD, designated RanBPM, was isolated with the two-hybrid method using Ran as a bait. Mouse and hamster RanBPM possessed a polypeptide identical to the human one. Furthermore, Saccharomyces cerevisiae was found to have a gene, YGL227w, the COOH-terminal half of which is 30% identical to RanBPM. Anti-RanBPM antibodies revealed that RanBPM was localized within the centrosome throughout the cell cycle. Overexpression of RanBPM produced multiple spots which were colocalized with gamma-tubulin and acted as ectopic microtubule nucleation sites, resulting in a reorganization of microtubule network. RanBPM cosedimented with the centrosomal fractions by sucrose- density gradient centrifugation. The formation of microtubule asters was inhibited not only by anti- RanBPM antibodies, but also by nonhydrolyzable GTP-Ran. Indeed, RanBPM specifically interacted with GTP-Ran in two-hybrid assay. The central part of asters stained by anti-RanBPM antibodies or by the mAb to gamma-tubulin was faded by the addition of GTPgammaS-Ran, but not by the addition of anti-RanBPM anti- bodies. These results provide evidence that the Ran-binding protein, RanBPM, is involved in microtubule nucleation, thereby suggesting that Ran regulates the centrosome through RanBPM.

Show MeSH
Related in: MedlinePlus