Our work provides unprecedented insight into the architecture of the centriole proximal region, which is key for a thorough understanding of the mechanisms governing centriole assembly.
Several mitotic regulators, including Cyclin B1/Cdk1, are present at centrosomes prior to mitosis onset, but it is unclear whether centrosomes promote mitotic entry in vivo. Here we developed a sensitive assay in C. elegans embryos for the temporal analysis of mitotic entry, in which the male and female pronuclei undergo asynchronous entry into mitosis when separated from one another. Using this assay, we found that centrosome integrity is necessary for timing mitotic entry. Centrosomes do not function in this instance through their ability to nucleate microtubules. Instead, centrosomes serve to focus the Aurora A kinase AIR-1, which is essential for timely mitotic entry. Furthermore, analysis of embryos in which centrosomes and pronuclei are detached from one another demonstrates that centrosomes are sufficient to promote mitosis onset. Together, our findings support a model in which centrosomes serve as integrative centers for mitotic regulators and thus trigger mitotic entry in a timely fashion.
Centrioles and basal bodies are essential for the formation of cilia, flagella, and centrosomes. They exhibit a characteristic ninefold symmetry imparted by a cartwheel thought to contain rings of SAS-6 proteins. We used cryoelectron tomography to investigate the architecture of the exceptionally long cartwheel of the flagellate Trichonympha. We found that the cartwheel is a stack of central rings that exhibit a vertical periodicity of 8.5 nanometers and is able to accommodate nine SAS-6 homodimers. The spokes that emanate from two such rings associate into a layer, with a vertical periodicity of 17 nanometers on the cartwheel margin. Thus, by using the power of biodiversity, we unveiled the architecture of the cartwheel at the root of the ninefold symmetry of centrioles and basal bodies.
Centrioles are evolutionary conserved organelles that give rise to cilia and flagella as well as centrosomes. Centrioles display a characteristic ninefold symmetry imposed by the spindle assembly abnormal protein 6 (SAS-6) family. SAS-6 from Chlamydomonas reinhardtii and Danio rerio was shown to form ninefold symmetric, ring-shaped oligomers in vitro that were similar to the cartwheels observed in vivo during early steps of centriole assembly in most species. Here, we report crystallographic and EM analyses showing that, instead, Caenorhabotis elegans SAS-6 self-assembles into a spiral arrangement. Remarkably, we find that this spiral arrangement is also consistent with ninefold symmetry, suggesting that two distinct SAS-6 oligomerization architectures can direct the same output symmetry. Sequence analysis suggests that SAS-6 spirals are restricted to specific nematodes. This oligomeric arrangement may provide a structural basis for the presence of a central tube instead of a cartwheel during centriole assembly in these species.X-ray crystallography | electron microscopy | structure | centriolar architecture | SAS-5
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