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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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Abnormal location of new BB formation in the NBD mutants. Log phase wild-type or MTT1::gtu1-A101G-HA cells grown at 15°C in MEPPS (0.5 μg/ml CdCl2) for 1–2 d were fixed and stained with anticentrin (green) and anti-K (red) antibodies. Pink arrows and white arrowheads indicate new BBs without anti-K staining and mature BBs with anti-K staining, respectively. (A) Stacks of both upper and lower halves of wild-type (a and b) and mutant cells (c and d) are shown. (e–g) Images from a, c, and d at higher magnification, respectively. Bars, 10 μm. (B) A Z-series showing thin sections of a MTT1::gtu1-A101G-HA mutant cell with internal BBs. Two focal planes (a and b) through internal sections of the cell are shown at higher magnification in c and d. Internal BBs were rarely seen in wild-type cells. (C) To study the spatial pattern of new BB formation, each cell was divided into four sectors (a), and the percentage of new BBs in each sector in each cell was calculated. Cells were then grouped into four categories based on which sector is the most active region for new BB assembly, and the number of cells in each category was determined. For the MTT1::gtu1-A101G-HA mutant, the new BBs inside or outside BB rows were calculated separately.
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fig4: Abnormal location of new BB formation in the NBD mutants. Log phase wild-type or MTT1::gtu1-A101G-HA cells grown at 15°C in MEPPS (0.5 μg/ml CdCl2) for 1–2 d were fixed and stained with anticentrin (green) and anti-K (red) antibodies. Pink arrows and white arrowheads indicate new BBs without anti-K staining and mature BBs with anti-K staining, respectively. (A) Stacks of both upper and lower halves of wild-type (a and b) and mutant cells (c and d) are shown. (e–g) Images from a, c, and d at higher magnification, respectively. Bars, 10 μm. (B) A Z-series showing thin sections of a MTT1::gtu1-A101G-HA mutant cell with internal BBs. Two focal planes (a and b) through internal sections of the cell are shown at higher magnification in c and d. Internal BBs were rarely seen in wild-type cells. (C) To study the spatial pattern of new BB formation, each cell was divided into four sectors (a), and the percentage of new BBs in each sector in each cell was calculated. Cells were then grouped into four categories based on which sector is the most active region for new BB assembly, and the number of cells in each category was determined. For the MTT1::gtu1-A101G-HA mutant, the new BBs inside or outside BB rows were calculated separately.

Mentions: We then examined whether the spatial pattern for the formation of new (immature) BBs was affected, which would strongly argue for ectopic BB formation. Antibodies against K antigens, which are proteins of unknown function that associate only with mature BBs (Williams et al., 1990), can be used to distinguish mature from newly formed BBs (Fig. 4 A, e; white arrowheads and pink arrows, respectively). In mutant cells, but not in wild-type cells, we observed immature BBs at abnormal positions not adjacent to a preexisting BBs (Fig. 4 A, f and g; pink arrows; similar results for MTT1:gtu1-T146V-HA are not depicted). Almost half of the BBs found near the cell surface but outside of rows in mutant cells were immature, and the ratio of immature to mature BBs not in rows was almost three times greater than the ratio within rows in the same cell (not depicted). These observations suggest that new BBs are forming ectopically in the cell cortex in mutant cells. We also detected ectopic formation of immature BBs in the interior cytoplasm beneath the cell surface (Fig. 4 B, a–d).


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)

Abnormal location of new BB formation in the NBD mutants. Log phase wild-type or MTT1::gtu1-A101G-HA cells grown at 15°C in MEPPS (0.5 μg/ml CdCl2) for 1–2 d were fixed and stained with anticentrin (green) and anti-K (red) antibodies. Pink arrows and white arrowheads indicate new BBs without anti-K staining and mature BBs with anti-K staining, respectively. (A) Stacks of both upper and lower halves of wild-type (a and b) and mutant cells (c and d) are shown. (e–g) Images from a, c, and d at higher magnification, respectively. Bars, 10 μm. (B) A Z-series showing thin sections of a MTT1::gtu1-A101G-HA mutant cell with internal BBs. Two focal planes (a and b) through internal sections of the cell are shown at higher magnification in c and d. Internal BBs were rarely seen in wild-type cells. (C) To study the spatial pattern of new BB formation, each cell was divided into four sectors (a), and the percentage of new BBs in each sector in each cell was calculated. Cells were then grouped into four categories based on which sector is the most active region for new BB assembly, and the number of cells in each category was determined. For the MTT1::gtu1-A101G-HA mutant, the new BBs inside or outside BB rows were calculated separately.
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Related In: Results  -  Collection

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fig4: Abnormal location of new BB formation in the NBD mutants. Log phase wild-type or MTT1::gtu1-A101G-HA cells grown at 15°C in MEPPS (0.5 μg/ml CdCl2) for 1–2 d were fixed and stained with anticentrin (green) and anti-K (red) antibodies. Pink arrows and white arrowheads indicate new BBs without anti-K staining and mature BBs with anti-K staining, respectively. (A) Stacks of both upper and lower halves of wild-type (a and b) and mutant cells (c and d) are shown. (e–g) Images from a, c, and d at higher magnification, respectively. Bars, 10 μm. (B) A Z-series showing thin sections of a MTT1::gtu1-A101G-HA mutant cell with internal BBs. Two focal planes (a and b) through internal sections of the cell are shown at higher magnification in c and d. Internal BBs were rarely seen in wild-type cells. (C) To study the spatial pattern of new BB formation, each cell was divided into four sectors (a), and the percentage of new BBs in each sector in each cell was calculated. Cells were then grouped into four categories based on which sector is the most active region for new BB assembly, and the number of cells in each category was determined. For the MTT1::gtu1-A101G-HA mutant, the new BBs inside or outside BB rows were calculated separately.
Mentions: We then examined whether the spatial pattern for the formation of new (immature) BBs was affected, which would strongly argue for ectopic BB formation. Antibodies against K antigens, which are proteins of unknown function that associate only with mature BBs (Williams et al., 1990), can be used to distinguish mature from newly formed BBs (Fig. 4 A, e; white arrowheads and pink arrows, respectively). In mutant cells, but not in wild-type cells, we observed immature BBs at abnormal positions not adjacent to a preexisting BBs (Fig. 4 A, f and g; pink arrows; similar results for MTT1:gtu1-T146V-HA are not depicted). Almost half of the BBs found near the cell surface but outside of rows in mutant cells were immature, and the ratio of immature to mature BBs not in rows was almost three times greater than the ratio within rows in the same cell (not depicted). These observations suggest that new BBs are forming ectopically in the cell cortex in mutant cells. We also detected ectopic formation of immature BBs in the interior cytoplasm beneath the cell surface (Fig. 4 B, a–d).

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