γ-Tubulin sequence comparison. Sequence comparison of the sequence of the T3 and T4 loops of γ-tubulin of T. thermophila and five other species with T. thermophila β-tubulin (BTU). GTU, γ-tubuin.

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.

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.

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.

The abnormal pattern of BBs in NBD mutants. Wild-type (WT), MTT1::gtu1-A101G-HA mutant, and T146V mutant strains (see Materials and methods for details) grown at 15 or 20°C in 0.5 μg/ml CdCl₂ for 1 or 4 d were fixed and stained with anticentrin (green) and anti-KF (red) antibodies. (A, a–d) Stacks of one half of each cell are shown. (e–h) Images at higher magnification. (a and e) Wild-type cell. (b, c, f, and g) Three types of defects in NBD mutants: (1) BBs with different orientations (arrows); (2) BBs not in rows (arrowheads); and (3) increased BB densities. The BB density (the number of BBs/250 μm²) is listed beneath the bottom panels. (d and h) As a control for the effects of cell enlargement, a ΔRFT2 mutant cell grown at 30°C for 3 d (these mutant cells do not grow at lower temperatures) shows incomplete division furrows without severe disruption of BB row organization or abnormal BB density. (B) Internal BBs in the cytoplasm of a wild-type cell and a mutant. A Z-series shows thin sections of a wild-type (a) and an MTT1::gtu1-A101G-HA cell (b) after 1 d at 15°C. (c and d) Images from the lettered panels (a and b) at higher magnification. Bars,10 μm.

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.

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.