We performed systematic mutagenesis of γ-tubulin and found that five regions on the protein's surface (plus end, minus end, H3 surface, ML surface, and COOH terminus) are important for γ-tubulin function in T. thermophila
, which is consistent with the studies performed in A. nidulans
and Schizosaccharomyces pombe
(Hendrickson et al., 2001
; Jung et al., 2001
; Vogel et al., 2001
). Many conditional mutants in these areas show defects in BB assembly, mimicking γ-tubulin depletion. Unlike the previous analyses of γ-tubulin mutations (Hendrickson et al., 2001
; Jung et al., 2001
; Vogel et al., 2001
), we also analyzed the NBD, showing that most mutations in the nucleotide-binding pocket are lethal, including T146G in the T4 loop (tubulin signature domain), suggesting an essential role for the NBD in γ-tubulin function in T. thermophila
In a further analysis of the function of the NBD, we studied the phenotype of two cold-sensitive mutant strains with a mutation (A101G) in the T3 loop (the second glycine-rich loop, mimicking the T4 loop) or in the T4 loop (T146V). Surprisingly, both mutations in the γ-tubulin gene conferred a novel phenotype with defects in the formation and organization of newly formed BBs but not in BB maturation, stability, or in γ-tubulin function in nuclear division.
One issue that arises is whether these mutations reflect a dominant gain of function or a recessive loss of function. Unfortunately, this is not easily determined in T. thermophila because it is not possible to create a stable diploid in which the mutant allele being tested is expressed in roughly the same amount as the wild-type allele. Although it is possible to create stable diploids in the germline micronucleus, the micronucleus is transcriptionally silent. It is impossible to create a stable diploid situation in the macronucleus, where genes are expressed, because it is polyploid and divides amitotically. Thus, chromosomes in the macronucleus do not segregate; they are distributed randomly to daughter cells in approximately (but not exactly) equal numbers. Therefore, even if one starts out with a heterozygote in the germline micronucleus, as soon as the macronucleus is formed from the micronucleus during the sexual process of conjugation, different cells have different numbers of each allele, and within ~50–100 fissions, all cells are completely homozygous in their macronuclei for one or the other allele (although they are still heterozygous in their micronucleus). Therefore, the conditions necessary to test for dominance cannot be achieved in T. thermophila.
We considered three explanations for the ectopic locations of new BBs: (1) deregulation of BB formation; (2) indirect effects of cell cycle arrest, as observed in some mammalian cells (Khodjakov et al., 2002
; Wong and Stearns, 2003
); and (3) indirect effects of structural/functional alterations in the individual BBs. Several lines of evidence suggest that the disruption of BB rows is caused by the deregulation of BB biogenesis.
First, we showed that BBs with random orientations could be detected at abnormal positions and that in some areas in mutant cells, the density of BBs is higher than that of wild-type cells, suggesting that new BB biogenesis is decoupled from the cell cycle. More importantly, we could detect newly formed BBs in abnormal positions, including deep in the cytoplasm, with random orientations, some of which were not adjacent to a mature BB. The anterior–posterior gradient of BB proliferation in mutant cells was also disrupted. In wild-type cells, the most active region of BB duplication is almost always localized to sector II, just posterior to the midline where the fission zone will be formed in the next division (Nanney, 1975
; Kaczanowski, 1978
), whereas this spatial pattern is disrupted in mutants. These data strongly suggest that new BBs were ectopically generated in these mutants.
Second, the appearance of BBs with different orientations at abnormal positions is growth dependent, suggesting that the stability of preexisting BBs was not severely affected. Therefore, the defects observed in the mutant cells were not results of the disassembly and delocalization of preexisting BBs from their original positions in BB rows caused by the mutant γ-tubulin protein, but instead resulted from the defects in BBs produced under restrictive conditions.
Third, we have presented evidence that the overproduction of BBs observed in the NBD mutants was not caused by cell cycle arrest at a stage when BBs are formed. Before cell division had ceased at the nonpermissive temperature, we observed an increased number of BBs with different orientations and outside of rows in dividing mutant cells exhibiting normal nuclear division and a normal fission furrow. In addition, when mutant strains were shifted from 15 to 30°C, they resumed growth within 5–7 h without detectable synchrony in micronuclear division. Moreover, very few mutant cells contained extra OAs or nuclei in any of the conditions tested. Finally, a strain with a control mutation (ΔRFT2) that blocks cytokinesis and produces enlarged cells did not show increased BB density or disorganization of BB rows. Thus, we conclude that overproduction of individual BBs in the NBD mutants is unlikely to result from a prolonged cell cycle stage.
Finally, the depletion–reinduction experiments showed that most BBs formed at restrictive temperature (15 or 20°C) were able to be assembled into BB rows and form cilia, and we did not detect any obvious defects in immunofluorescent studies with antibodies that detect either BBs or BB-associated structures. These results indicate that both BB maturation and stability in the mutant cells at the restrictive condition are not severely affected, arguing that the mutations do not affect the function of BBs.
Therefore, we conclude that mutations in the NBD of the T. thermophila γ-tubulin gene lead to deregulation of BB formation, and, as a result, mutant cells are able to make BBs with random orientations at ectopic locations where BB assembly normally is not allowed. This process of unregulated BB duplication likely causes the organization of cortical rows to be disrupted.
In T. thermophila
, γ-tubulin is required to nucleate and maintain centriolar MTs and MTs that are required for micro- and macronuclear division (Shang et al., 2002a
). Strikingly, the only severe defects we observed in the two NBD mutant cells described in this study are overproduction and ectopic formation of new BBs. Thus, the γ-tubulin NBD is specifically required for both temporal and spatial restriction of BB formation, and the activity of γ-tubulin in the initiation of BB/centriole formation can be uncoupled from its MT-nucleating activity.
In the templated pathway of BB formation, daughter BBs/centrioles are always formed perpendicular to old ones (for review see Beisson and Wright, 2003
). In T. thermophila
, the spatial regulation is further restricted so new BBs are only formed just anterior to old ones (Nanney, 1975
; Kaczanowski, 1978
). In the NBD mutants, this spatial restriction is severely compromised. One possibility is that without the normal γ-tubulin NBD, new BBs can form de novo and in a more spatially unrestricted manner relative to global cell coordinates. Alternatively, but not necessarily mutually exclusive, new BBs may still be formed near old ones but at unconstrained positions. We favor the first hypothesis because we observed many new BBs both outside of BB rows and underneath the cell surface, not adjacent to preexisting BBs.
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 (, 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 (, 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
Figure 7. 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 (more ...)
In conclusion, we have demonstrated that mutations in the NBD of γ-tubulin lead to nontemplated formation of BBs and uncoupling of BB biogenesis from the cell cycle. We have shown that this is not likely to be a secondary effect caused by cell cycle arrest, supporting the hypothesis that γ-tubulin is directly involved in intiating centiole/BB assembly and that its nucleation activity is normally suppressed by an unidentified negative regulating complex through interaction with its NBD. Future studies will hopefully identify the components of this complex. Unfortunately, methods are not yet available in T. thermophila for performing screens for allele-specific suppressors, and the unique genetic code (TAA/G = gln) makes traditional two-hybrid approaches difficult.