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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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Cellular localization of RanBPM. (A) Nonsynchronized cultures of  MRC5 cells were fixed and  permeabilized as described in  Materials and Methods. Cells  were double stained with the  mAb to α-tubulin (green) and  with the affinity-purified anti-RanBPM antibodies (red).  Red and green staining patterns were superimposed by  electronic image processing  (superimposition). The representative figures are shown.  Bars, 5 μm. (B and C) Nonsynchronized cultures of  MRC5 cells were fixed and  doubly stained with affinity-purified anti-RanBPM antibodies (red) and the mAb to  γ-tubulin (green) (B), or with  affinity-purified anti–γ-tubulin antibodies (red) and the  mAb to α-tubulin (green)  (C). Red and green staining  patterns were superimposed  by electronic image processing (superimposition). Cells  within the same visual field  are demonstrated by antibody staining and phase–contrast. Bars, 10 μm.
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Figure 2: Cellular localization of RanBPM. (A) Nonsynchronized cultures of MRC5 cells were fixed and permeabilized as described in Materials and Methods. Cells were double stained with the mAb to α-tubulin (green) and with the affinity-purified anti-RanBPM antibodies (red). Red and green staining patterns were superimposed by electronic image processing (superimposition). The representative figures are shown. Bars, 5 μm. (B and C) Nonsynchronized cultures of MRC5 cells were fixed and doubly stained with affinity-purified anti-RanBPM antibodies (red) and the mAb to γ-tubulin (green) (B), or with affinity-purified anti–γ-tubulin antibodies (red) and the mAb to α-tubulin (green) (C). Red and green staining patterns were superimposed by electronic image processing (superimposition). Cells within the same visual field are demonstrated by antibody staining and phase–contrast. Bars, 10 μm.

Mentions: Immunoblotting analysis using the affinity-purified anti-RanBPM antibodies showed that whereas several protein bands were recognized in the extracts of HeLa cells, only a single band of 55 kD was recognized in the extracts of MRC5 cells. To determine the cellular localization of RanBPM, cultures of MRC5 cells were doubly stained with the affinity-purified anti-RanBPM antibodies (red) and the mAb to α-tubulin (green) (Fig. 2 A). When both staining patterns overlapped, the microtubule was found to be nucleated from the matrix which was stained with the affinity-purified anti-RanBPM antibodies. These results suggested that the protein encoded by the B8 clone was localized within the centrosome, and thereby RanBPM stands for Ran-binding protein in MTOC.


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)

Cellular localization of RanBPM. (A) Nonsynchronized cultures of  MRC5 cells were fixed and  permeabilized as described in  Materials and Methods. Cells  were double stained with the  mAb to α-tubulin (green) and  with the affinity-purified anti-RanBPM antibodies (red).  Red and green staining patterns were superimposed by  electronic image processing  (superimposition). The representative figures are shown.  Bars, 5 μm. (B and C) Nonsynchronized cultures of  MRC5 cells were fixed and  doubly stained with affinity-purified anti-RanBPM antibodies (red) and the mAb to  γ-tubulin (green) (B), or with  affinity-purified anti–γ-tubulin antibodies (red) and the  mAb to α-tubulin (green)  (C). Red and green staining  patterns were superimposed  by electronic image processing (superimposition). Cells  within the same visual field  are demonstrated by antibody staining and phase–contrast. Bars, 10 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132962&req=5

Figure 2: Cellular localization of RanBPM. (A) Nonsynchronized cultures of MRC5 cells were fixed and permeabilized as described in Materials and Methods. Cells were double stained with the mAb to α-tubulin (green) and with the affinity-purified anti-RanBPM antibodies (red). Red and green staining patterns were superimposed by electronic image processing (superimposition). The representative figures are shown. Bars, 5 μm. (B and C) Nonsynchronized cultures of MRC5 cells were fixed and doubly stained with affinity-purified anti-RanBPM antibodies (red) and the mAb to γ-tubulin (green) (B), or with affinity-purified anti–γ-tubulin antibodies (red) and the mAb to α-tubulin (green) (C). Red and green staining patterns were superimposed by electronic image processing (superimposition). Cells within the same visual field are demonstrated by antibody staining and phase–contrast. Bars, 10 μm.
Mentions: Immunoblotting analysis using the affinity-purified anti-RanBPM antibodies showed that whereas several protein bands were recognized in the extracts of HeLa cells, only a single band of 55 kD was recognized in the extracts of MRC5 cells. To determine the cellular localization of RanBPM, cultures of MRC5 cells were doubly stained with the affinity-purified anti-RanBPM antibodies (red) and the mAb to α-tubulin (green) (Fig. 2 A). When both staining patterns overlapped, the microtubule was found to be nucleated from the matrix which was stained with the affinity-purified anti-RanBPM antibodies. These results suggested that the protein encoded by the B8 clone was localized within the centrosome, and thereby RanBPM stands for Ran-binding protein in MTOC.

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