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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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Mutations in the NBD, but not K358AK359A on the inside surface, showed BB overproduction. Log phase MTT1::GTU1-HA (WT), MTT::gtu1-K358AK359A-HA, and MTT1::gtu1-A101G-HA grown at 30°C were shifted to 15°C in MEPPS containing 0.5 μg/ml CdCl2 for the indicated period and were stained with anticentrin antibodies for BB morphology and DAPI for nuclear morphology. (A, a) Normal BB morphology in (wild type) MTT1::GTU1-HA cells; centrin antibody stains BBs in the oral apparatus (OA) and somatic basal body (sBB). (A, b) Failure of mutant MTT1::gtu1-K358AK359A-HA cells to duplicate BBs (loss of BB). (A, c) BB overproduction in MTT1::gtu1-A101G-HA cells is indicated by the increased density of BBs in some BB rows and OA. (B) Western blot probed with anti-HA antibodies to determine γ-tubulin levels in the three strains as shown.
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fig1: Mutations in the NBD, but not K358AK359A on the inside surface, showed BB overproduction. Log phase MTT1::GTU1-HA (WT), MTT::gtu1-K358AK359A-HA, and MTT1::gtu1-A101G-HA grown at 30°C were shifted to 15°C in MEPPS containing 0.5 μg/ml CdCl2 for the indicated period and were stained with anticentrin antibodies for BB morphology and DAPI for nuclear morphology. (A, a) Normal BB morphology in (wild type) MTT1::GTU1-HA cells; centrin antibody stains BBs in the oral apparatus (OA) and somatic basal body (sBB). (A, b) Failure of mutant MTT1::gtu1-K358AK359A-HA cells to duplicate BBs (loss of BB). (A, c) BB overproduction in MTT1::gtu1-A101G-HA cells is indicated by the increased density of BBs in some BB rows and OA. (B) Western blot probed with anti-HA antibodies to determine γ-tubulin levels in the three strains as shown.

Mentions: We first analyzed the phenotypes of mutations in the five regions homologous to those studied previously in fungal γ-tubulins (Table I; Hendrickson et al., 2001; Jung et al., 2001) at permissive (30°C) and nonpermissive (40, 15, or 20°C) temperatures. Normal T. thermophila cells contain large number of BBs, including those found in the OA at the anterior end and in rows of somatic BBs oriented in the long axis of the cell (Fig. 1 A, a). Most mutations in the five regions were either lethal or yielded cold-sensitive mutants that showed BB phenotypes similar to those described for γ-tubulin depletion in T. thermophila (Table I and Fig. 1 A, b; Shang et al., 2002a), including strong staining of γ-tubulin on the interphase macronuclear envelope and defects in BB duplication and BB stability (Shang et al., 2002a; unpublished data). None showed significant phenotypes specifically at a high temperature (40°C). Cells with mutations in these regions lost their OA and most somatic BBs at 15°C. Centrin staining of the remaining somatic BBs also showed an abnormal dispersed pattern rather than punctuate dots. Thus, mutations in these regions yielded hypomorphic phenotypes, indicating they are important for folding, stability, or other general functions of γ-tubulin in T. thermophila (Hendrickson et al., 2001; Jung et al., 2001). These mutants were not further studied.


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)

Mutations in the NBD, but not K358AK359A on the inside surface, showed BB overproduction. Log phase MTT1::GTU1-HA (WT), MTT::gtu1-K358AK359A-HA, and MTT1::gtu1-A101G-HA grown at 30°C were shifted to 15°C in MEPPS containing 0.5 μg/ml CdCl2 for the indicated period and were stained with anticentrin antibodies for BB morphology and DAPI for nuclear morphology. (A, a) Normal BB morphology in (wild type) MTT1::GTU1-HA cells; centrin antibody stains BBs in the oral apparatus (OA) and somatic basal body (sBB). (A, b) Failure of mutant MTT1::gtu1-K358AK359A-HA cells to duplicate BBs (loss of BB). (A, c) BB overproduction in MTT1::gtu1-A101G-HA cells is indicated by the increased density of BBs in some BB rows and OA. (B) Western blot probed with anti-HA antibodies to determine γ-tubulin levels in the three strains as shown.
© Copyright Policy
Related In: Results  -  Collection

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fig1: Mutations in the NBD, but not K358AK359A on the inside surface, showed BB overproduction. Log phase MTT1::GTU1-HA (WT), MTT::gtu1-K358AK359A-HA, and MTT1::gtu1-A101G-HA grown at 30°C were shifted to 15°C in MEPPS containing 0.5 μg/ml CdCl2 for the indicated period and were stained with anticentrin antibodies for BB morphology and DAPI for nuclear morphology. (A, a) Normal BB morphology in (wild type) MTT1::GTU1-HA cells; centrin antibody stains BBs in the oral apparatus (OA) and somatic basal body (sBB). (A, b) Failure of mutant MTT1::gtu1-K358AK359A-HA cells to duplicate BBs (loss of BB). (A, c) BB overproduction in MTT1::gtu1-A101G-HA cells is indicated by the increased density of BBs in some BB rows and OA. (B) Western blot probed with anti-HA antibodies to determine γ-tubulin levels in the three strains as shown.
Mentions: We first analyzed the phenotypes of mutations in the five regions homologous to those studied previously in fungal γ-tubulins (Table I; Hendrickson et al., 2001; Jung et al., 2001) at permissive (30°C) and nonpermissive (40, 15, or 20°C) temperatures. Normal T. thermophila cells contain large number of BBs, including those found in the OA at the anterior end and in rows of somatic BBs oriented in the long axis of the cell (Fig. 1 A, a). Most mutations in the five regions were either lethal or yielded cold-sensitive mutants that showed BB phenotypes similar to those described for γ-tubulin depletion in T. thermophila (Table I and Fig. 1 A, b; Shang et al., 2002a), including strong staining of γ-tubulin on the interphase macronuclear envelope and defects in BB duplication and BB stability (Shang et al., 2002a; unpublished data). None showed significant phenotypes specifically at a high temperature (40°C). Cells with mutations in these regions lost their OA and most somatic BBs at 15°C. Centrin staining of the remaining somatic BBs also showed an abnormal dispersed pattern rather than punctuate dots. Thus, mutations in these regions yielded hypomorphic phenotypes, indicating they are important for folding, stability, or other general functions of γ-tubulin in T. thermophila (Hendrickson et al., 2001; Jung et al., 2001). These mutants were not further studied.

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