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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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NBD mutant cells can regenerate BBs without severe structural/functional defects at the restrictive temperature. The γ-tubulin–depleted wild-type or MTT1::gtu1-A101G-HA cells were shifted to 15°C in MEPPS containing 0.5 μg/ml CdCl2 for the indicated times, fixed, and stained with anticentrin antibody to visualize and count BBs (A and C). (A) The percentage of recovering cells with BB rows. (B) Immunofluorescence staining with anti-KF (red) and anticentrin (green) shows one fully recovered wild-type cell (inset) and five MTT1::gtu1-A101G-HA mutant cells showing a high density of BBs in some areas and BB rows in different orientations. Bar, 10 μm. (C) The number of BBs/cell in wild-type and MTT1::gtu1-A101G-HA cells was counted 29 d after recovery. Cells with different number of BBs were grouped into seven categories, and the percentage of cells in each category was determined. (D) Immunofluorescence staining with antipolyglutamic acid antibody (green) and anticentrin (red) shows that many BBs synthesized at 15°C have nucleated cilia.
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fig6: NBD mutant cells can regenerate BBs without severe structural/functional defects at the restrictive temperature. The γ-tubulin–depleted wild-type or MTT1::gtu1-A101G-HA cells were shifted to 15°C in MEPPS containing 0.5 μg/ml CdCl2 for the indicated times, fixed, and stained with anticentrin antibody to visualize and count BBs (A and C). (A) The percentage of recovering cells with BB rows. (B) Immunofluorescence staining with anti-KF (red) and anticentrin (green) shows one fully recovered wild-type cell (inset) and five MTT1::gtu1-A101G-HA mutant cells showing a high density of BBs in some areas and BB rows in different orientations. Bar, 10 μm. (C) The number of BBs/cell in wild-type and MTT1::gtu1-A101G-HA cells was counted 29 d after recovery. Cells with different number of BBs were grouped into seven categories, and the percentage of cells in each category was determined. (D) Immunofluorescence staining with antipolyglutamic acid antibody (green) and anticentrin (red) shows that many BBs synthesized at 15°C have nucleated cilia.

Mentions: To determine whether mutant BBs formed at the restrictive temperature have structural or functional defects that prevent them from maturing and forming rows, we performed depletion–reinduction experiments. Because the γ-tubulin genes in both the two mutants and the wild-type strain described in this study are under control of the MTT1 promoter, their expression requires cadmium (Shang et al., 2002a,b). Removal of cadmium at the permissive temperature resulted in the depletion of γ-tubulin and the disassembly of most preexisting BBs in all three strains. When the expression of γ-tubulin was restored by adding cadmium at the restrictive temperature, wild-type cells recovered normal cortical structure. Although mutants failed to fully recover, most of the new BBs formed at 15°C could be assembled into short BB rows at a similar rate as wild-type cells (Fig. 6 A) and were associated with the two mature BB-associated structures, KFs (Fig. 6 B) and K antigens (not depicted). By 29 d, many mutant cells had assembled more BBs than the control strain (Fig. 6 C) and showed increased BB densities in some rows, suggesting that in the mutant cells, the rate of BB formation is uncoupled from (and exceeds) the rate of cell growth. Staining with an antipolyglutamic acid antibody (Shang et al., 2002a) showed that most of the mutant BBs that assembled at 15°C contained polyglutamylated tubulins and were associated with cilia (Fig. 6 D), which require functional BBs for their formation and maintenance.


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)

NBD mutant cells can regenerate BBs without severe structural/functional defects at the restrictive temperature. The γ-tubulin–depleted wild-type or MTT1::gtu1-A101G-HA cells were shifted to 15°C in MEPPS containing 0.5 μg/ml CdCl2 for the indicated times, fixed, and stained with anticentrin antibody to visualize and count BBs (A and C). (A) The percentage of recovering cells with BB rows. (B) Immunofluorescence staining with anti-KF (red) and anticentrin (green) shows one fully recovered wild-type cell (inset) and five MTT1::gtu1-A101G-HA mutant cells showing a high density of BBs in some areas and BB rows in different orientations. Bar, 10 μm. (C) The number of BBs/cell in wild-type and MTT1::gtu1-A101G-HA cells was counted 29 d after recovery. Cells with different number of BBs were grouped into seven categories, and the percentage of cells in each category was determined. (D) Immunofluorescence staining with antipolyglutamic acid antibody (green) and anticentrin (red) shows that many BBs synthesized at 15°C have nucleated cilia.
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fig6: NBD mutant cells can regenerate BBs without severe structural/functional defects at the restrictive temperature. The γ-tubulin–depleted wild-type or MTT1::gtu1-A101G-HA cells were shifted to 15°C in MEPPS containing 0.5 μg/ml CdCl2 for the indicated times, fixed, and stained with anticentrin antibody to visualize and count BBs (A and C). (A) The percentage of recovering cells with BB rows. (B) Immunofluorescence staining with anti-KF (red) and anticentrin (green) shows one fully recovered wild-type cell (inset) and five MTT1::gtu1-A101G-HA mutant cells showing a high density of BBs in some areas and BB rows in different orientations. Bar, 10 μm. (C) The number of BBs/cell in wild-type and MTT1::gtu1-A101G-HA cells was counted 29 d after recovery. Cells with different number of BBs were grouped into seven categories, and the percentage of cells in each category was determined. (D) Immunofluorescence staining with antipolyglutamic acid antibody (green) and anticentrin (red) shows that many BBs synthesized at 15°C have nucleated cilia.
Mentions: To determine whether mutant BBs formed at the restrictive temperature have structural or functional defects that prevent them from maturing and forming rows, we performed depletion–reinduction experiments. Because the γ-tubulin genes in both the two mutants and the wild-type strain described in this study are under control of the MTT1 promoter, their expression requires cadmium (Shang et al., 2002a,b). Removal of cadmium at the permissive temperature resulted in the depletion of γ-tubulin and the disassembly of most preexisting BBs in all three strains. When the expression of γ-tubulin was restored by adding cadmium at the restrictive temperature, wild-type cells recovered normal cortical structure. Although mutants failed to fully recover, most of the new BBs formed at 15°C could be assembled into short BB rows at a similar rate as wild-type cells (Fig. 6 A) and were associated with the two mature BB-associated structures, KFs (Fig. 6 B) and K antigens (not depicted). By 29 d, many mutant cells had assembled more BBs than the control strain (Fig. 6 C) and showed increased BB densities in some rows, suggesting that in the mutant cells, the rate of BB formation is uncoupled from (and exceeds) the rate of cell growth. Staining with an antipolyglutamic acid antibody (Shang et al., 2002a) showed that most of the mutant BBs that assembled at 15°C contained polyglutamylated tubulins and were associated with cilia (Fig. 6 D), which require functional BBs for their formation and maintenance.

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