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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 formation of new BBs in sector III of dividing mutant cells. Both MTT1::GTU1-HA (a) and MTT1::gtu1-A101G-HA strains (b) were shifted from 30 to 15°C and stained with anticentrin (green) and anti-KF (red) antibodies. A dividing cell with a fission zone from each strain is shown. (c and d) Images at higher magnification. Sector II, the active zone for BB duplication, is normally posterior to the fission zone. In MTT1::gtu1-A101G-HA strains (d), the arrow shows BBs with abnormal orientation, and arrowheads show BBs in abnormal positions. Bars, 10 μm.
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fig5: Abnormal formation of new BBs in sector III of dividing mutant cells. Both MTT1::GTU1-HA (a) and MTT1::gtu1-A101G-HA strains (b) were shifted from 30 to 15°C and stained with anticentrin (green) and anti-KF (red) antibodies. A dividing cell with a fission zone from each strain is shown. (c and d) Images at higher magnification. Sector II, the active zone for BB duplication, is normally posterior to the fission zone. In MTT1::gtu1-A101G-HA strains (d), the arrow shows BBs with abnormal orientation, and arrowheads show BBs in abnormal positions. Bars, 10 μm.

Mentions: Abnormal BB phenotypes were detected in mutant cells within 1 d after they were shifted to the restrictive temperature, whereas the cells continued dividing for 3 d. Before cell division had ceased at the nonpermissive temperature, in some dividing mutant cells that exhibited normal nuclear division and a normal fission furrow, we observed increased numbers of BBs that were misoriented and were located outside of rows in sector III (Fig. 5; similar results for MTT1:gtu1-T146V-HA are not depicted). Also, when mutant strains were released from 15 to 30°C, they resumed growth within 5–7 h (not depicted) without detectable synchrony in micronuclear division (not depicted), indicating that the mutant cells were not blocked at a specific stage of the cell cycle.


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 formation of new BBs in sector III of dividing mutant cells. Both MTT1::GTU1-HA (a) and MTT1::gtu1-A101G-HA strains (b) were shifted from 30 to 15°C and stained with anticentrin (green) and anti-KF (red) antibodies. A dividing cell with a fission zone from each strain is shown. (c and d) Images at higher magnification. Sector II, the active zone for BB duplication, is normally posterior to the fission zone. In MTT1::gtu1-A101G-HA strains (d), the arrow shows BBs with abnormal orientation, and arrowheads show BBs in abnormal positions. Bars, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171320&req=5

fig5: Abnormal formation of new BBs in sector III of dividing mutant cells. Both MTT1::GTU1-HA (a) and MTT1::gtu1-A101G-HA strains (b) were shifted from 30 to 15°C and stained with anticentrin (green) and anti-KF (red) antibodies. A dividing cell with a fission zone from each strain is shown. (c and d) Images at higher magnification. Sector II, the active zone for BB duplication, is normally posterior to the fission zone. In MTT1::gtu1-A101G-HA strains (d), the arrow shows BBs with abnormal orientation, and arrowheads show BBs in abnormal positions. Bars, 10 μm.
Mentions: Abnormal BB phenotypes were detected in mutant cells within 1 d after they were shifted to the restrictive temperature, whereas the cells continued dividing for 3 d. Before cell division had ceased at the nonpermissive temperature, in some dividing mutant cells that exhibited normal nuclear division and a normal fission furrow, we observed increased numbers of BBs that were misoriented and were located outside of rows in sector III (Fig. 5; similar results for MTT1:gtu1-T146V-HA are not depicted). Also, when mutant strains were released from 15 to 30°C, they resumed growth within 5–7 h (not depicted) without detectable synchrony in micronuclear division (not depicted), indicating that the mutant cells were not blocked at a specific stage of the cell cycle.

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