Mechanisms of HsSAS-6 assembly promoting centriole formation in human cells.

SAS-6 proteins are thought to impart the ninefold symmetry of centrioles, but the mechanisms by which their assembly occurs within cells remain elusive. In this paper, we provide evidence that the N-terminal, coiled-coil, and C-terminal domains of HsSAS-6 are each required for procentriole formation in human cells. Moreover, the coiled coil is necessary and sufficient to mediate HsSAS-6 centrosomal targeting. High-resolution imaging reveals that GFP-tagged HsSAS-6 variants localize in a torus around the base of the parental centriole before S phase, perhaps indicative of an initial loading platform. Moreover, fluorescence recovery after photobleaching analysis demonstrates that HsSAS-6 is immobilized progressively at centrosomes during cell cycle progression. Using fluorescence correlation spectroscopy and three-dimensional stochastic optical reconstruction microscopy, we uncover that HsSAS-6 is present in the cytoplasm primarily as a homodimer and that its oligomerization into a ninefold symmetrical ring occurs at centrioles. Together, our findings lead us to propose a mechanism whereby HsSAS-6 homodimers are targeted to centrosomes where the local environment and high concentration of HsSAS-6 promote oligomerization, thus initiating procentriole formation.

Native architecture of the centriole proximal region reveals features underlying its 9-fold radial symmetry.

BACKGROUND: Centrioles are cylindrical microtubule-based structures whose assembly is critical for the formation of cilia, flagella, and centrosomes. The centriole proximal region harbors a cartwheel that dictates the 9-fold symmetry of centrioles. Although the cartwheel architecture has been recently analyzed, how it connects to the peripheral microtubules is not understood. More generally, a high-resolution view of the proximal region of the centriole is lacking, thus limiting understanding of the underlying assembly mechanisms. RESULTS: We report the complete architecture of the Trichonympha centriole proximal region using cryotomography. The resulting 3D map reveals several features, including additional densities in the cartwheel that exhibit a 9-fold symmetrical arrangement, as well as the structure of the Pinhead and the A-C linker that connect to microtubules. Moreover, we uncover striking chiral features that might impart directionality to the entire centriole. Furthermore, we identify Trichonympha SAS-6 and demonstrate that it localizes to the cartwheel in vivo. CONCLUSIONS: 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.

Caenorhabditis elegans centriolar protein SAS-6 forms a spiral that is consistent with imparting a ninefold symmetry.

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.

Cartwheel architecture of Trichonympha basal body.

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.

The nucleoporin Nup205/NPP-3 is lost near centrosomes at mitotic onset and can modulate the timing of this process in Caenorhabditis elegans embryos.

Regulation of mitosis in time and space is critical for proper cell division. We conducted an RNA interference-based modifier screen to identify novel regulators of mitosis in Caenorhabditis elegans embryos. Of particular interest, this screen revealed that the Nup205 nucleoporin NPP-3 can negatively modulate the timing of mitotic onset. Furthermore, we discovered that NPP-3 and nucleoporins that are associated with it are lost from the nuclear envelope (NE) in the vicinity of centrosomes at the onset of mitosis. We demonstrate that centrosomes are both necessary and sufficient for NPP-3 local loss, which also requires the activity of the Aurora-A kinase AIR-1. Our findings taken together support a model in which centrosomes and AIR-1 promote timely onset of mitosis by locally removing NPP-3 and associated nucleoporins from the NE.

Centrosomes promote timely mitotic entry in C. elegans embryos.

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.

Promotion of importin alpha-mediated nuclear import by the phosphorylation-dependent binding of cargo protein to 14-3-3.

14-3-3 proteins are phosphoserine/threonine-binding proteins that play important roles in many regulatory processes, including intracellular protein targeting. 14-3-3 proteins can anchor target proteins in the cytoplasm and in the nucleus or can mediate their nuclear export. So far, no role for 14-3-3 in mediating nuclear import has been described. There is also mounting evidence that nuclear import is regulated by the phosphorylation of cargo proteins, but the underlying mechanism remains elusive. Myopodin is a dual-compartment, actin-bundling protein that functions as a tumor suppressor in human bladder cancer. In muscle cells, myopodin redistributes between the nucleus and the cytoplasm in a differentiation-dependent and stress-induced fashion. We show that importin alpha binding and the subsequent nuclear import of myopodin are regulated by the serine/threonine phosphorylation-dependent binding of myopodin to 14-3-3. These results establish a novel paradigm for the promotion of nuclear import by 14-3-3 binding. They provide a molecular explanation for the phosphorylation-dependent nuclear import of nuclear localization signal-containing cargo proteins.

Importin alpha associates with membranes and participates in nuclear envelope assembly in vitro.

Importin alpha is well known as an adaptor that functions with Importin beta in the nuclear import of proteins containing specific nuclear localization signals (NLSs). We show here that either an excess or a lack of Importin alpha blocks nuclear envelope (NE) assembly in vitro, and our data suggest that soluble Importin alpha functions in NE assembly in conjunction with NLS-containing partner proteins. Surprisingly, a significant proportion of Importin alpha is found to fractionate with Xenopus egg membranes. We demonstrate that membrane association of Importin alpha is regulated by phosphorylation. Using mutant forms of Importin alpha that either do not bind membranes or are not released from them by phosphorylation, we provide evidence that membrane-associated Importin alpha is required for NE formation. Unlike other functions of Importin alpha, this membrane-associated activity does not require interaction with NLS proteins.