To study the possible involvement of HsCen3p in centrosome duplication, we performed overexpression of wild-type and mutant forms of HsCen3p in human cultured cells, but did not succeed in disturbing the centrosome duplication cycle in transient transfection experiments. The overexpression of HsCen3p was found to be consistently less efficient than overexpression of HsCen2p, suggesting that either the level of HsCen3p is more tightly regulated than that of HsCen2p in human cells or that the half life of the two proteins are quite different. We observed that inactivation of the fourth EF-hand prevents centriole localization of the protein. However, part of this mutant protein can associate with Triton X-100 insoluble structures. A long-term effect of high dosage of HsCen3p was suggested by the fact that we could not establish stable cell lines expressing HsCen3p, whereas we could easily do it with the two other human centrin genes. We reasoned that a high dosage of HsCen3p could have a deleterious effect only after one or two centrosome duplication cycles, a possibility which was difficult to test in culture cells. Thus, we turned to the Xenopus
embryo, which we previously used to study HsCen2p function (Paoletti et al. 1996
). In this rapidly dividing system, HsCen2p and HsCen3p led to completely different effects, although undercleavage was observed in both cases: undercleaved blastomeres produced by injection of HsCen2p contained numerous asters, indicating that the centrosome duplication process itself was not prevented. Thus, HsCen2p could disturb either cytokinesis itself or any upstream event, for example, the timing of centrosome duplication or its coupling with cytokinesis. On the contrary, HsCen3p is shown in this work to impair centrosome duplication. As a consequence, cleavage is inhibited. Our results do not exclude, however, that HsCen3p also impairs events downstream of centrosome reproduction, including the cleavage process itself. This could explain that a few uncleaved blastomeres contained more than two microtubule asters. Injection of RNA encoding the mutant HsCen3p-D147,149,151A protein, which is unable to localize to the centrosome, also impairs centrosome duplication, suggesting that HsCen3p acts by interacting with a cytoplasmic XCen3p-binding protein.
We observed that HsCEN3
could not complement S
strains bearing temperature-sensitive mutations in CDC31
or a complete deletion of CDC31
. On the contrary, HsCen3p inhibited cell growth and SPB duplication, whereas the two other human centrin proteins were without effect. We have shown that HsCen3p interacts with Kar1p, a protein which is also involved in SPB duplication (Biggins and Rose 1994
), whereas the other known Cdc31p-binding protein, Kic1p, is involved in cell wall integrity (Sullivan et al. 1998
). We also found that HsCen2p, which did not impair cell growth when overexpressed in yeast, is also able to interact with Kar1p as previously described in vitro (Geier et al. 1996
). This suggests that HsCen3p disturbs SPB duplication by titrating other Cdc31p-binding proteins. This was further confirmed: HsCen3p still inhibits SPB duplication in a yeast strain expressing the cdc31-16
allele that can grow in the absence of Kar1p. It is noteworthy that, in the conditions we used to demonstrate the interaction between HsCen3p and Kar1p, we could not detect an interaction between Cdc31p and Kar1p. This result suggests that Kar1p has a higher affinity for HsCen3p and for HsCen2p than for Cdc31p or, more likely, that the functional Cdc31p/Kar1p complex, which is part of the half bridge, is not soluble in the conditions we used. No SPB localization has been detected for HsCen3p, making it possible that HsCen3p sequesters Cdc31p-binding proteins in the cytoplasm. The existence of complexes between HsCen3p and Cdc31p-binding protein(s) is in favor of a functional conservation between HsCen3p and Cdc31p.
Altogether, our data strongly argue in favor of the existence of two functionally distinct centrin families (see ): a first one implicated in centrosome duplication, to which Cdc31p and HsCen3p belong; and a second family that participates in other cell division events, such as centrosome segregation or cytokinesis, and which includes centrin from C
and HsCen2p (Paoletti et al. 1996
). The single centrin gene of the budding yeast might be able to fulfill both functions. It has been shown that, in addition to SPB duplication, Cdc31p regulates the activity of Kic1p, a kinase involved in cell integrity and in cell separation (Sullivan et al. 1998
Recently, several studies have shown that cyclin-CDKs are required for driving centrosome duplication in animals. During embryonic cell cycles, cyclin E-CDK2 activity is required for centrosome reproduction (Hinchcliffe et al. 1999
; Lacey et al. 1999
), whereas cyclin A-CDK2 is also required in somatic cells (Matsumoto et al. 1999
; Meraldi et al. 1999
). Altogether, these data suggest that the cell cycle machinery indeed regulates centrosome reproduction, coupling it with the cell cycle. However, the centrosomal targets of cyclins-CDK2 are unknown. It is possible that these kinases transcriptionally regulate genes involved in centrosome duplication. Identification of genes coding for centrosomal proteins regulated by cyclin-CDKs will be a crucial step in understanding centrosome duplication regulation.
The intriguing possibility that γ-tubulin, a protein involved in microtubule nucleation and stability, is required for basal body assembly in Paramecium
suggests that controlling microtubule dynamics might also regulate centriole/basal body duplication and probably that the regulation of centriolar microtubule assembly shares common steps with cytoplasmic microtubule nucleation (Ruiz et al. 1999
). Centrin 3 is the first centriole-associated protein, actually concentrated in the distal lumen of each centriole and in the early procentriole bud, to be shown to participate in the initiation of centrosome duplication in animals. Identification of proteins interacting with Cen3p in a Ca2+
-dependent manner should be critical for further study of the regulation of the centrosome duplication. As HsCen3p blocks yeast SPB and frog centrosome duplication most likely by competing with Cdc31p and with Xenopus
Cen3p for their physiological targets, the two experimental systems used in this study provide valuable tools to identify new proteins involved in SPB or centrosome duplication.