Figure 1.
Distribution of α- and γ-tubulin in the C. elegans centrosome.
A. Immunofluorescence images (projections of confocal stacks) of a wild-type embryo (left column) and a tbg-1(RNAi) embryo (right column) showing α-tubulin (red), γ-tubulin (green), and DNA (blue). B. Line scans from single confocal planes over centrosome regions as indicated in (A) used for measurements of pixel intensity. C. Linescan intensity plots of fluorescence intensity for α-tubulin (red) and γ-tubulin (green) along the marked lines presented in (B). In contrast to the tbg-1(RNAi) embryo, the concentration α-tubulin is locally reduced in the wild-type centrosome. Bar: 10 µm in A.
Figure 2.
Structural organization of the mitotic centrosome in the one-cell wild-type and tbg1(RNAi) embryo.
A. Tomographic slices of centrosomes containing centriole pairs (c) in one-celled metaphase embryos at early metaphase (wild-type: left column; tbg-1(RNAi): right column) showing that the central core (dashed line) in the centrosome is lost after depletion of γ-tubulin. B. Modeling of centrioles (central tube in orange, centriolar microtubules in blue) and spindle microtubules (red) from the centrosome as shown in A. The microtubules in the tbg-1(RNAi) centrosome appear to traverse through and encircle the centrosome in contrast to the radial arrangement seen in wild-type embryos. The number of pole-proximal ends (white spheres) is reduced in the tbg-1(RNAi) embryo. Bars: 200 nm in A and B.
Table 1.
Comparison of wild type embryos with TBG-1 and SPD-5 depletion phenotypes.
Figure 3.
Distribution and morphology of pole-proximal microtubule ends in mitotic centrosomes.
A. Gallery of pole-proximal ends (wild-type: left column; tbg-1(RNAi): right column). Electron tomography reveals closed- and open-end morphologies. B. 3-D modeling of closed (purple spheres) and open (yellow spheres) microtubule ends. C. The ratio of closed/open pole-proximal ends is reversed in γ-tubulin compromised embryos. D. Distribution of closed microtubule pole-proximal ends. Although reduced in number, the closed pole-proximal ends in tbg-1(RNAi) embryos occupy a similar distance from the centrosome center as wild type. Bars: 50 nm in A; 200 nm in B.
Figure 4.
Effect of γ-tubulin-RNAi on centriole ultrastructure.
A. Tomographic slice showing centriolar microtubules extending beyond the distal ends of the two centrioles (arrows). B–C. 3-D models of centrioles showing that both mother and daughter centrioles can contain elongated centriolar microtubules (B), however some tomograms contained only single centrioles (C). D. Length of central tubes from tbg-1(RNAi) centrosomes showing double or single centrioles. Bars: 200 nm A–C.
Figure 5.
Effect of spd-5(RNAi) on centrosome structure and microtubule end morphology.
A. Live-cell imaging of a wild-type and spd-5(RNAi) embryo expressing β-tubulin::GFP. B. Imaging of TBG-1::GFP in spd-5(RNAi) embryos. C. Western blot. γ-tubulin is present in the SPD-5-compromised embryo. A non-specific band from the anti-SPD-5 antiserum served as a loading control (l.c.). D. Gallery of closed (left images) and open (right images) pole-proximal ends in spd-5(RNAi) embryos. E. Modeling of centrioles (central tube in orange, centriolar microtubules in blue) and spindle microtubules. Compared to wild type, the number of pole-proximal ends (white spheres) is reduced and these ends are distributed near the centrioles within the centrosome core. The centriolar microtubules are extended. F. 3-D modeling of closed (purple spheres) and open (yellow spheres) microtubule ends. G. The closed microtubule pole-proximal ends are closer to the centrosome center compared to wild type. H. Percentage of closed vs. open pole proximal ends. Bars: 10 mm A and C; 50 nm D; 200 nm E and F.
Figure 6.
Model explaining the relationship between centrosomal microtubule organization and centriole morphology.
A–B. Centriole pairs in spd-5(RNAi) embryos with variable lengths of the central tube of daughter centrioles. C. Centrosome region with three centrioles at one pole. Although it is unclear how one extra centriole could arise within a single cell cycle, the loss of SPD-5 may cause excessive movement of mother and daughter centrioles during the duplication process. If a daughter is dislodged at an early stage of this process, perhaps the same mother centriole can support the assembly of a second daughter. D. Quantification of central tube length in wild-type versus spd-5(RNAi) embryos. E. Model explaining the effect of TBG-1 and SPD-5 depletion on the structural organization of the centrosome. In wild type, the PCM component SPD-5 and γ-tubulin might contribute to a zone of exclusion visible as a ring-shaped area of pole-proximal microtubule ends around centrioles that limits microtubule polymerization and physically prevents movement of microtubule polymers into the centrosome core. Centriolar microtubule assembly occurs normally during late prophase, in a SAS-4 dependent manner. The zone of exclusion might trap SAS-4 within this central core, which could specifically promote the assembly of centriolar microtubules in an environment expected to be otherwise non-conducive to microtubule polymerization. In tbg-1(RNAi) or spd-5(RNAi) embryos, microtubule polymerization occurs near the centrioles, exchange with cytoplasmic SAS-4 becomes possible, and centriolar microtubules over-extend. Microtubule numbers are greatly reduced in spd-5(RNAi) but less affected in tbg-1(RNAi), where the majority of microtubule ends near the centrosome exhibit an open, rather than a closed morphology. Bars: 200 nm A–C.