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Mutational analyses reveal a novel function of the nucleotide-binding domain of gamma-tubulin in the regulation of basal body biogenesis.

Shang Y, Tsao CC, Gorovsky MA - J. Cell Biol. (2005)

Bottom Line: These results, coupled with previous studies (Dammermann, A., T.McEwen, G.Khodjakov. 2005.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Rochester, Rochester, NY 14627, USA.

ABSTRACT
We have used in vitro mutagenesis and gene replacement to study the function of the nucleotide-binding domain (NBD) of gamma-tubulin in Tetrahymena thermophila. In this study, we show that the NBD has an essential function and that point mutations in two conserved residues lead to over-production and mislocalization of basal body (BB) assembly. These results, coupled with previous studies (Dammermann, A., T. Muller-Reichert, L. Pelletier, B. Habermann, A. Desai, and K. Oegema. 2004. Dev. Cell. 7:815-829; La Terra, S., C.N. English, P. Hergert, B.F. McEwen, G. Sluder, and A. Khodjakov. 2005. J. Cell Biol. 168:713-722), suggest that to achieve the precise temporal and spatial regulation of BB/centriole assembly, the initiation activity of gamma-tubulin is normally suppressed by a negative regulatory mechanism that acts through its NBD.

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The abnormal pattern of BBs in NBD mutants. Wild-type (WT), MTT1::gtu1-A101G-HA mutant, and T146V mutant strains (see Materials and methods for details) grown at 15 or 20°C in 0.5 μg/ml CdCl2 for 1 or 4 d were fixed and stained with anticentrin (green) and anti-KF (red) antibodies. (A, a–d) Stacks of one half of each cell are shown. (e–h) Images at higher magnification. (a and e) Wild-type cell. (b, c, f, and g) Three types of defects in NBD mutants: (1) BBs with different orientations (arrows); (2) BBs not in rows (arrowheads); and (3) increased BB densities. The BB density (the number of BBs/250 μm2) is listed beneath the bottom panels. (d and h) As a control for the effects of cell enlargement, a ΔRFT2 mutant cell grown at 30°C for 3 d (these mutant cells do not grow at lower temperatures) shows incomplete division furrows without severe disruption of BB row organization or abnormal BB density. (B) Internal BBs in the cytoplasm of a wild-type cell and a mutant. A Z-series shows thin sections of a wild-type (a) and an MTT1::gtu1-A101G-HA cell (b) after 1 d at 15°C. (c and d) Images from the lettered panels (a and b) at higher magnification. Bars,10 μm.
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fig3: The abnormal pattern of BBs in NBD mutants. Wild-type (WT), MTT1::gtu1-A101G-HA mutant, and T146V mutant strains (see Materials and methods for details) grown at 15 or 20°C in 0.5 μg/ml CdCl2 for 1 or 4 d were fixed and stained with anticentrin (green) and anti-KF (red) antibodies. (A, a–d) Stacks of one half of each cell are shown. (e–h) Images at higher magnification. (a and e) Wild-type cell. (b, c, f, and g) Three types of defects in NBD mutants: (1) BBs with different orientations (arrows); (2) BBs not in rows (arrowheads); and (3) increased BB densities. The BB density (the number of BBs/250 μm2) is listed beneath the bottom panels. (d and h) As a control for the effects of cell enlargement, a ΔRFT2 mutant cell grown at 30°C for 3 d (these mutant cells do not grow at lower temperatures) shows incomplete division furrows without severe disruption of BB row organization or abnormal BB density. (B) Internal BBs in the cytoplasm of a wild-type cell and a mutant. A Z-series shows thin sections of a wild-type (a) and an MTT1::gtu1-A101G-HA cell (b) after 1 d at 15°C. (c and d) Images from the lettered panels (a and b) at higher magnification. Bars,10 μm.

Mentions: In T. thermophila, the orientation and position of individual mature BBs can be detected easily by antibodies against kinetodesmal fibers (KFs), which are nonmicrotubular structures that associate only with mature somatic BBs (Jerka-Dziadosz et al., 1995; Shang et al., 2002a). In wild-type cells, KFs are oriented parallel to the longitudinal axis of the cell, running from posterior to anterior on the same side of all somatic BBs (Fig. 3 A, a and e). Within 1 d of cold treatment, before most of the mutant cells had ceased dividing, BBs were observed with KFs pointing in random directions (Fig. 3 A, f and g; arrows), indicating a failure to orient properly. In addition, some BBs were abnormally positioned outside of rows and were not adjacent to a preexisting BB (Fig. 3 A, f and g; arrowheads). Internal BBs in the cytoplasm beneath the cell surface were also detected (Fig. 3 B; similar results for MTT1:gtu1-T146V-HA mutants are not depicted). The existence of internal BBs was confirmed by transmission EM (not depicted). Again, the density of the somatic BBs in some areas of the mutant cells is higher compared with the wild-type strain (Fig. 3 A, bottom), suggesting that these mutant cells generate more BBs than wild-type controls. Therefore, the two mutations in the NBD of γ-tubulin lead to defects in BB orientation, position, and density.


Mutational analyses reveal a novel function of the nucleotide-binding domain of gamma-tubulin in the regulation of basal body biogenesis.

Shang Y, Tsao CC, Gorovsky MA - J. Cell Biol. (2005)

The abnormal pattern of BBs in NBD mutants. Wild-type (WT), MTT1::gtu1-A101G-HA mutant, and T146V mutant strains (see Materials and methods for details) grown at 15 or 20°C in 0.5 μg/ml CdCl2 for 1 or 4 d were fixed and stained with anticentrin (green) and anti-KF (red) antibodies. (A, a–d) Stacks of one half of each cell are shown. (e–h) Images at higher magnification. (a and e) Wild-type cell. (b, c, f, and g) Three types of defects in NBD mutants: (1) BBs with different orientations (arrows); (2) BBs not in rows (arrowheads); and (3) increased BB densities. The BB density (the number of BBs/250 μm2) is listed beneath the bottom panels. (d and h) As a control for the effects of cell enlargement, a ΔRFT2 mutant cell grown at 30°C for 3 d (these mutant cells do not grow at lower temperatures) shows incomplete division furrows without severe disruption of BB row organization or abnormal BB density. (B) Internal BBs in the cytoplasm of a wild-type cell and a mutant. A Z-series shows thin sections of a wild-type (a) and an MTT1::gtu1-A101G-HA cell (b) after 1 d at 15°C. (c and d) Images from the lettered panels (a and b) at higher magnification. Bars,10 μm.
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fig3: The abnormal pattern of BBs in NBD mutants. Wild-type (WT), MTT1::gtu1-A101G-HA mutant, and T146V mutant strains (see Materials and methods for details) grown at 15 or 20°C in 0.5 μg/ml CdCl2 for 1 or 4 d were fixed and stained with anticentrin (green) and anti-KF (red) antibodies. (A, a–d) Stacks of one half of each cell are shown. (e–h) Images at higher magnification. (a and e) Wild-type cell. (b, c, f, and g) Three types of defects in NBD mutants: (1) BBs with different orientations (arrows); (2) BBs not in rows (arrowheads); and (3) increased BB densities. The BB density (the number of BBs/250 μm2) is listed beneath the bottom panels. (d and h) As a control for the effects of cell enlargement, a ΔRFT2 mutant cell grown at 30°C for 3 d (these mutant cells do not grow at lower temperatures) shows incomplete division furrows without severe disruption of BB row organization or abnormal BB density. (B) Internal BBs in the cytoplasm of a wild-type cell and a mutant. A Z-series shows thin sections of a wild-type (a) and an MTT1::gtu1-A101G-HA cell (b) after 1 d at 15°C. (c and d) Images from the lettered panels (a and b) at higher magnification. Bars,10 μm.
Mentions: In T. thermophila, the orientation and position of individual mature BBs can be detected easily by antibodies against kinetodesmal fibers (KFs), which are nonmicrotubular structures that associate only with mature somatic BBs (Jerka-Dziadosz et al., 1995; Shang et al., 2002a). In wild-type cells, KFs are oriented parallel to the longitudinal axis of the cell, running from posterior to anterior on the same side of all somatic BBs (Fig. 3 A, a and e). Within 1 d of cold treatment, before most of the mutant cells had ceased dividing, BBs were observed with KFs pointing in random directions (Fig. 3 A, f and g; arrows), indicating a failure to orient properly. In addition, some BBs were abnormally positioned outside of rows and were not adjacent to a preexisting BB (Fig. 3 A, f and g; arrowheads). Internal BBs in the cytoplasm beneath the cell surface were also detected (Fig. 3 B; similar results for MTT1:gtu1-T146V-HA mutants are not depicted). The existence of internal BBs was confirmed by transmission EM (not depicted). Again, the density of the somatic BBs in some areas of the mutant cells is higher compared with the wild-type strain (Fig. 3 A, bottom), suggesting that these mutant cells generate more BBs than wild-type controls. Therefore, the two mutations in the NBD of γ-tubulin lead to defects in BB orientation, position, and density.

Bottom Line: These results, coupled with previous studies (Dammermann, A., T.McEwen, G.Khodjakov. 2005.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Rochester, Rochester, NY 14627, USA.

ABSTRACT
We have used in vitro mutagenesis and gene replacement to study the function of the nucleotide-binding domain (NBD) of gamma-tubulin in Tetrahymena thermophila. In this study, we show that the NBD has an essential function and that point mutations in two conserved residues lead to over-production and mislocalization of basal body (BB) assembly. These results, coupled with previous studies (Dammermann, A., T. Muller-Reichert, L. Pelletier, B. Habermann, A. Desai, and K. Oegema. 2004. Dev. Cell. 7:815-829; La Terra, S., C.N. English, P. Hergert, B.F. McEwen, G. Sluder, and A. Khodjakov. 2005. J. Cell Biol. 168:713-722), suggest that to achieve the precise temporal and spatial regulation of BB/centriole assembly, the initiation activity of gamma-tubulin is normally suppressed by a negative regulatory mechanism that acts through its NBD.

Show MeSH
Related in: MedlinePlus