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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.

Show MeSH
A model for the role of γ-tubulin in centriole/BB biogenesis. We propose that the initiation activity of γ-tubulin is inhibited by a mechanism acting through the NBD (step 0). Nonexclusive possibilities include mechanisms that monitor or are sensitive to the GTP/GDP ratio in γ-tubulin or posttranslational modifications (that might be mimicked by NBD mutations) that regulate the association–dissociation of inhibitory complexes with the NBD. During initiation, an activating signal either inactivates the inhibitors (step 1a) or acts on γ-tubulin (step 1b) to convert it from an inactive to a licensed form that is competent to initiate duplication. The precise site of the active γ-tubulin complex depends on whether cells contain a preexisting BB/centriole. The precentriole particles containing inactive γ-tubulin will be degraded. Activation and recruitment of other BB/centriole components are then initiated. γ-Tubulin is also known to be involved in MT nucleation and long-term maintenance of BBs. γ-Tubulin indicates an inactive γ-tubulin complex; γ-tubulin* indicates an active form. MT, microtubule.
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fig7: A model for the role of γ-tubulin in centriole/BB biogenesis. We propose that the initiation activity of γ-tubulin is inhibited by a mechanism acting through the NBD (step 0). Nonexclusive possibilities include mechanisms that monitor or are sensitive to the GTP/GDP ratio in γ-tubulin or posttranslational modifications (that might be mimicked by NBD mutations) that regulate the association–dissociation of inhibitory complexes with the NBD. During initiation, an activating signal either inactivates the inhibitors (step 1a) or acts on γ-tubulin (step 1b) to convert it from an inactive to a licensed form that is competent to initiate duplication. The precise site of the active γ-tubulin complex depends on whether cells contain a preexisting BB/centriole. The precentriole particles containing inactive γ-tubulin will be degraded. Activation and recruitment of other BB/centriole components are then initiated. γ-Tubulin is also known to be involved in MT nucleation and long-term maintenance of BBs. γ-Tubulin indicates an inactive γ-tubulin complex; γ-tubulin* indicates an active form. MT, microtubule.

Mentions: Mechanistically, we propose that to prevent overproduction and ectopic nontemplated formation of BBs, the initiation of BB assembly must normally be suppressed by an inhibitory process that acts through the NBD (Fig. 7, step 0). In mammalian tissue culture cells, both templated and de novo centriole biogenesis begin with the formation of multiple precentriolar particles containing centrin and γ-tubulin, which can form multiple centrioles (de novo) or can coalesce to form a single centriole (templated) depending on whether the cell contains a preexisting centriole (La Terra et al., 2005). A single centriole can prevent the de novo formation of centrioles, indicating that there is a trans-acting signal that inhibits de novo formation. Our studies suggest that the NBD of γ-tubulin is the cis-acting target of this signal. Because normal templated assembly and experimentally induced de novo assembly of centrioles both occur in the same cell cycle stage (S phase; Ruiz et al., 1999; Marshall et al., 2001; Khodjakov et al., 2002; La Terra et al., 2005) and γ-tubulin is involved in both pathways (Khodjakov et al., 2002; Shang et al., 2002a; Suh et al., 2002), this inhibitory machinery is likely to suppress both pathways, and the initiation of both pathways probably requires the same trans-activators (Hinchcliffe and Sluder, 2001; Matsumoto and Maller, 2002) to release the inhibition and enable (license) the γ-tubulin complex to function (Fig. 7, steps 1a and 1b). Additional cell cycle–regulated steps also are required for newly formed centrioles to mature into MTOCs (La Terra et al., 2005).


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)

A model for the role of γ-tubulin in centriole/BB biogenesis. We propose that the initiation activity of γ-tubulin is inhibited by a mechanism acting through the NBD (step 0). Nonexclusive possibilities include mechanisms that monitor or are sensitive to the GTP/GDP ratio in γ-tubulin or posttranslational modifications (that might be mimicked by NBD mutations) that regulate the association–dissociation of inhibitory complexes with the NBD. During initiation, an activating signal either inactivates the inhibitors (step 1a) or acts on γ-tubulin (step 1b) to convert it from an inactive to a licensed form that is competent to initiate duplication. The precise site of the active γ-tubulin complex depends on whether cells contain a preexisting BB/centriole. The precentriole particles containing inactive γ-tubulin will be degraded. Activation and recruitment of other BB/centriole components are then initiated. γ-Tubulin is also known to be involved in MT nucleation and long-term maintenance of BBs. γ-Tubulin indicates an inactive γ-tubulin complex; γ-tubulin* indicates an active form. MT, microtubule.
© Copyright Policy
Related In: Results  -  Collection

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

fig7: A model for the role of γ-tubulin in centriole/BB biogenesis. We propose that the initiation activity of γ-tubulin is inhibited by a mechanism acting through the NBD (step 0). Nonexclusive possibilities include mechanisms that monitor or are sensitive to the GTP/GDP ratio in γ-tubulin or posttranslational modifications (that might be mimicked by NBD mutations) that regulate the association–dissociation of inhibitory complexes with the NBD. During initiation, an activating signal either inactivates the inhibitors (step 1a) or acts on γ-tubulin (step 1b) to convert it from an inactive to a licensed form that is competent to initiate duplication. The precise site of the active γ-tubulin complex depends on whether cells contain a preexisting BB/centriole. The precentriole particles containing inactive γ-tubulin will be degraded. Activation and recruitment of other BB/centriole components are then initiated. γ-Tubulin is also known to be involved in MT nucleation and long-term maintenance of BBs. γ-Tubulin indicates an inactive γ-tubulin complex; γ-tubulin* indicates an active form. MT, microtubule.
Mentions: Mechanistically, we propose that to prevent overproduction and ectopic nontemplated formation of BBs, the initiation of BB assembly must normally be suppressed by an inhibitory process that acts through the NBD (Fig. 7, step 0). In mammalian tissue culture cells, both templated and de novo centriole biogenesis begin with the formation of multiple precentriolar particles containing centrin and γ-tubulin, which can form multiple centrioles (de novo) or can coalesce to form a single centriole (templated) depending on whether the cell contains a preexisting centriole (La Terra et al., 2005). A single centriole can prevent the de novo formation of centrioles, indicating that there is a trans-acting signal that inhibits de novo formation. Our studies suggest that the NBD of γ-tubulin is the cis-acting target of this signal. Because normal templated assembly and experimentally induced de novo assembly of centrioles both occur in the same cell cycle stage (S phase; Ruiz et al., 1999; Marshall et al., 2001; Khodjakov et al., 2002; La Terra et al., 2005) and γ-tubulin is involved in both pathways (Khodjakov et al., 2002; Shang et al., 2002a; Suh et al., 2002), this inhibitory machinery is likely to suppress both pathways, and the initiation of both pathways probably requires the same trans-activators (Hinchcliffe and Sluder, 2001; Matsumoto and Maller, 2002) to release the inhibition and enable (license) the γ-tubulin complex to function (Fig. 7, steps 1a and 1b). Additional cell cycle–regulated steps also are required for newly formed centrioles to mature into MTOCs (La Terra et al., 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.

Show MeSH