Search

Binding of STIL activates Plk4, and the subsequent phosphorylation of STIL by Plk4 primes the binding of STIL to SAS6 to promote centriole assembly., Centriole duplication occurs once per cell cycle in order to maintain control of centrosome number and ensure genome integrity. Polo-like kinase 4 (Plk4) is a master regulator of centriole biogenesis, but how its activity is regulated to control centriole assembly is unclear. Here we used gene editing in human cells to create a chemical genetic system in which endogenous Plk4 can be specifically inhibited using a cell-permeable ATP analogue. Using this system, we demonstrate that STIL localization to the centriole requires continued Plk4 activity. Most importantly, we show that direct binding of STIL activates Plk4 by promoting self-phosphorylation of the activation loop of the kinase. Plk4 subsequently phosphorylates STIL to promote centriole assembly in two steps. First, Plk4 activity promotes the recruitment of STIL to the centriole. Second, Plk4 primes the direct binding of STIL to the C terminus of SAS6. Our findings uncover a molecular basis for the timing of Plk4 activation through the cell cycle–regulated accumulation of STIL.

Poc1 shores up basal bodies to support cilia formation in Tetrahymena thermophila, zebrafish, and humans; Poc1 depletion causes phenotypes commonly seen in ciliopathies., Centrioles are the foundation for centrosome and cilia formation. The biogenesis of centrioles is initiated by an assembly mechanism that first synthesizes the ninefold symmetrical cartwheel and subsequently leads to a stable cylindrical microtubule scaffold that is capable of withstanding microtubule-based forces generated by centrosomes and cilia. We report that the conserved WD40 repeat domain–containing cartwheel protein Poc1 is required for the structural maintenance of centrioles in Tetrahymena thermophila. Furthermore, human Poc1B is required for primary ciliogenesis, and in zebrafish, DrPoc1B knockdown causes ciliary defects and morphological phenotypes consistent with human ciliopathies. T. thermophila Poc1 exhibits a protein incorporation profile commonly associated with structural centriole components in which the majority of Poc1 is stably incorporated during new centriole assembly. A second dynamic population assembles throughout the cell cycle. Our experiments identify novel roles for Poc1 in centriole stability and ciliogenesis.

It has been reported that nontransformed mammalian cells become arrested during G1 in the absence of centrioles (Hinchcliffe, E., F. Miller, M. Cham, A. Khodjakov, and G. Sluder. 2001. Science. 291:1547–1550). Here, we show that removal of resident centrioles (by laser ablation or needle microsurgery) does not impede cell cycle progression in HeLa cells. HeLa cells born without centrosomes, later, assemble a variable number of centrioles de novo. Centriole assembly begins with the formation of small centrin aggregates that appear during the S phase. These, initially amorphous “precentrioles” become morphologically recognizable centrioles before mitosis. De novo–assembled centrioles mature (i.e., gain abilities to organize microtubules and replicate) in the next cell cycle. This maturation is not simply a time-dependent phenomenon, because de novo–formed centrioles do not mature if they are assembled in S phase–arrested cells. By selectively ablating only one centriole at a time, we find that the presence of a single centriole inhibits the assembly of additional centrioles, indicating that centrioles have an activity that suppresses the de novo pathway.

Cep120, a protein involved in maintenance of neural progenitor cells, is required for centriole duplication in cycling cells and for centriole amplification in tracheal epithelial cells., Centrioles form the core of the centrosome in animal cells and function as basal bodies that nucleate and anchor cilia at the plasma membrane. In this paper, we report that Cep120 (Ccdc100), a protein previously shown to be involved in maintaining the neural progenitor pool in mouse brain, is associated with centriole structure and function. Cep120 is up-regulated sevenfold during differentiation of mouse tracheal epithelial cells (MTECs) and localizes to basal bodies. Cep120 localizes preferentially to the daughter centriole in cycling cells, and this asymmetry between mother and daughter centrioles is relieved coincident with new centriole assembly. Photobleaching recovery analysis identifies two pools of Cep120, differing in their halftime at the centriole. We find that Cep120 is required for centriole duplication in cycling cells, centriole amplification in MTECs, and centriole overduplication in S phase–arrested cells. We propose that Cep120 is required for centriole assembly and that the observed defect in neuronal migration might derive from a defect in this process.

Plk4 phosphorylates itself in trans to prevent accumulation and self-limit kinase activity, which may be important for regulating centriole duplication., Accurate control of the number of centrosomes, the major microtubule-organizing centers of animal cells, is critical for the maintenance of genome integrity. Abnormalities in centrosome number can promote errors in spindle formation that lead to subsequent chromosome missegregation, and extra centrosomes are found in many cancers. Centrosomes are comprised of a pair of centrioles surrounded by amorphous pericentriolar material, and centrosome duplication is controlled by centriole replication. Polo-like kinase 4 (Plk4) plays a key role in initiating centriole duplication, and overexpression of Plk4 promotes centriole overduplication and the formation of extra centrosomes. Using chemical genetics, we show that kinase-active Plk4 is inherently unstable and targeted for degradation. Plk4 is shown to multiply self-phosphorylate within a 24–amino acid phosphodegron. Phosphorylation of multiple sites is required for Plk4 instability, indicating a requirement for a threshold level of Plk4 kinase activity to promote its own destruction. We propose that kinase-mediated, autoregulated instability of Plk4 self-limits Plk4 activity so as to prevent centrosome amplification.

Centrioles are surrounded by pericentriolar material (PCM), which is proposed to promote new centriole assembly by concentrating γ-tubulin. Here, we quantitatively monitor new centriole assembly in living Caenorhabditis elegans embryos, focusing on the conserved components SAS-4 and SAS-6. We show that SAS-4 and SAS-6 are coordinately recruited to the site of new centriole assembly and reach their maximum levels during S phase. Centriolar SAS-6 is subsequently reduced by a mechanism intrinsic to the early assembly pathway that does not require progression into mitosis. Centriolar SAS-4 remains in dynamic equilibrium with the cytoplasmic pool until late prophase, when it is stably incorporated in a step that requires γ-tubulin and microtubule assembly. These results indicate that γ-tubulin in the PCM stabilizes the nascent daughter centriole by promoting microtubule addition to its outer wall. Such a mechanism may help restrict new centriole assembly to the vicinity of preexisting parent centrioles that recruit PCM.

Ciliated epithelial cells have the unique ability to generate hundreds of centrioles during differentiation. We used centrosomal proteins as molecular markers in cultured mouse tracheal epithelial cells to understand this process. Most centrosomal proteins were up-regulated early in ciliogenesis, initially appearing in cytoplasmic foci and then incorporated into centrioles. Three candidate proteins were further characterized. The centrosomal component SAS-6 localized to basal bodies and the proximal region of the ciliary axoneme, and depletion of SAS-6 prevented centriole assembly. The intraflagellar transport component polaris localized to nascent centrioles before incorporation into cilia, and depletion of polaris blocked axoneme formation. The centriolar satellite component PCM-1 colocalized with centrosomal components in cytoplasmic granules surrounding nascent centrioles. Interfering with PCM-1 reduced the amount of centrosomal proteins at basal bodies but did not prevent centriole assembly. This system will help determine the mechanism of centriole formation in mammalian cells and how the limitation on centriole duplication is overcome in ciliated epithelial cells.

We have generated several stable cell lines expressing GFP-labeled centrin. This fusion protein becomes concentrated in the lumen of both centrioles, making them clearly visible in the living cell. Time-lapse fluorescence microscopy reveals that the centriole pair inherited after mitosis splits during or just after telophase. At this time the mother centriole remains near the cell center while the daughter migrates extensively throughout the cytoplasm. This differential behavior is not related to the presence of a nucleus because it is also observed in enucleated cells. The characteristic motions of the daughter centriole persist in the absence of microtubules (Mts). or actin, but are arrested when both Mts and actin filaments are disrupted. As the centrioles replicate at the G1/S transition the movements exhibited by the original daughter become progressively attenuated, and by the onset of mitosis its behavior is indistinguishable from that of the mother centriole. While both centrioles possess associated gamma-tubulin, and nucleate similar number of Mts in Mt repolymerization experiments. during G1 and S only the mother centriole is located at the focus of the Mt array. A model, based on differences in Mt anchoring and release by the mother and daughter centrioles, is proposed to explain these results.

In interphase Chinese hamster ovary (CHO) cells, the centrosome is attached to the nucleus very firmly. This nuclear-centrosome complex is isolated as a coherent structure by lysis and extraction of cells with Triton X-100 in a low ionic strength medium. Under these conditions, the ultrastructure of the centrioles attached to the nucleus can be discerned by electron microscopy of whole-mount preparations. The structural changes of the centrioles as a function of the cell cycle were monitored by this technique. Specifically, centriolar profiles were placed into six categories according to their orientation and the length ratio of daughter and parent centrioles. The proportion of centrioles in each category was plotted as a frequency histogram. The morphological changes in the centriole cycle were characterized by three distinguishable events: nucleation, elongation, and disorientation. The progress of centrioles through these stages was determined in synchronous populations of cells starting from S or M phase, in cells inhibited in DNA synthesis by addition of thymidine, and in cytoplasts. The results provide a quantitative description of the events of the centriole cycle. They also show that, in complete cells, nucleation, elongation, and disorientation are not dependent upon DNA synthesis. However, in cytoplasts, although elongation and disorientation occur as in normal cells, nucleation is blocked. Procentriole formation appeared to be inhibited by the removal of the nucleus. We suggest that coordination of centriole replication and nuclear replication may depend upon a signal arising from the nucleus.