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Polo-like kinase 4 (Plk4) is a key regulator of centriole duplication, but the mechanism underlying its recruitment to mammalian centrioles is not understood. In flies, Plk4 recruitment depends on Asterless, whereas nematodes rely on a distinct protein, Spd-2. Here, we have explored the roles of two homologous mammalian proteins, Cep152 and Cep192, in the centriole recruitment of human Plk4. We demonstrate that Cep192 plays a key role in centrosome recruitment of both Cep152 and Plk4. Double-depletion of Cep192 and Cep152 completely abolishes Plk4 binding to centrioles as well as centriole duplication, indicating that the two proteins cooperate. Most importantly, we show that Cep192 binds Plk4 through an N-terminal extension that is specific to the largest isoform. The Plk4 binding regions of Cep192 and Cep152 (residues 190-240 and 1-46, respectively) are rich in negatively charged amino acids, suggesting that Plk4 localization to centrioles depends on electrostatic interactions with the positively charged polo-box domain. We conclude that cooperation between Cep192 and Cep152 is crucial for centriole recruitment of Plk4 and centriole duplication during the cell cycle.

Control of centriole number is crucial for genome stability and ciliogenesis. Here, we characterize the role of human STIL, a protein that displays distant sequence similarity to the centriole duplication factors Ana2 in Drosophila and SAS-5 in Caenorhabditis elegans. Using RNA interference, we show that STIL is required for centriole duplication in human cells. Conversely, overexpression of STIL triggers the near-simultaneous formation of multiple daughter centrioles surrounding each mother, which is highly reminiscent of the phenotype produced by overexpression of the polo-like kinase PLK4 or the spindle assembly abnormal protein 6 homolog (SAS-6). We further show, by fluorescence and immunoelectron microscopy, that STIL is recruited to nascent daughter centrioles at the onset of centriole duplication and degraded, in an APC/C(Cdc20-Cdh1)-dependent manner, upon passage through mitosis. We did not detect a stable complex between STIL and SAS-6, but the two proteins resemble each other with regard to both localization and cell cycle control of expression. Thus, STIL cooperates with SAS-6 and PLK4 in the control of centriole number and represents a key centriole duplication factor in human cells.

Centrioles function as the major components of centrosomes, which organize microtubule (MT) arrays in proliferating cells, and as basal bodies for primary cilia formation in quiescent cells. Centrioles and basal bodies are structurally similar, barrel-shaped organelles composed of MTs. In proliferating cells, two new centrioles, termed procentrioles, form during the S phase of the cell cycle in close proximity to the proximal ends of the two preexisting parental centrioles, often at a near-orthogonal angle. Considerable progress has been made toward understanding the biogenesis of centrioles, but the mechanisms that determine their lengths remain unknown. Here we show that overexpression of the centriolar protein CPAP in human cells enhances the accumulation of centriolar tubulin, leading to centrioles of strikingly increased length. Consistent with earlier work, we also find that elongated MT structures can be induced by depletion of the distal end-capping protein CP110 from centrioles. Importantly, though, these structures differ from genuine primary cilia. We thus propose that CPAP and CP110 play antagonistic roles in determining the extent of tubulin addition during centriole elongation, thereby controlling the length of newly formed centrioles.

The centrosome duplicates during the cell cycle but functions as a single microtubule-organising centre until shortly before mitosis. This raises the question of how centrosome cohesion is maintained throughout interphase. One dynamic model proposes that parental centrioles are held together through centriole-associated, entangling filaments. Central to this model are C-Nap1, a putative centriolar docking protein and rootletin, a fibrous component. Here we identify two novel proteins, Cep68 and Cep215, as required for centrosome cohesion. Similar to rootletin, Cep68 decorates fibres emanating from the proximal ends of centrioles and dissociates from centrosomes during mitosis. Furthermore, Cep68 and rootletin depend both on each other and on C-Nap1 for centriole association. Unlike rootletin, overexpression of Cep68 does not induce extensive fibre formation, but Cep68 is readily recruited to ectopic rootletin fibres. These data suggest that Cep68 cooperates with rootletin and C-Nap1 in centrosome cohesion. By contrast, Cep215 associates with centrosomes throughout the cell cycle and does not appear to interact with Cep68, rootletin or C-Nap1. Instead, our data suggest that Cep215 functionally interacts with pericentrin, suggesting that both proteins influence centrosome cohesion through an indirect mechanism related to cytoskeletal dynamics.

Primary cilia (PC) function as microtubule-based sensory antennae projecting from the surface of many eukaryotic cells. They play important roles in mechano- and chemosensory perception and their dysfunction is implicated in developmental disorders and severe diseases. The basal body that functions in PC assembly is derived from the mature centriole, a component of the centrosome. Through a small interfering RNA screen we found several centrosomal proteins (Ceps) to be involved in PC formation. One newly identified protein, Cep164, was indispensable for PC formation and hence characterized in detail. By immunogold electron microscopy, Cep164 could be localized to the distal appendages of mature centrioles. In contrast to ninein and Cep170, two components of subdistal appendages, Cep164 persisted at centrioles throughout mitosis. Moreover, the localizations of Cep164 and ninein/Cep170 were mutually independent during interphase. These data implicate distal appendages in PC formation and identify Cep164 as an excellent marker for these structures.

We show that overexpression of Polo-like kinase 4 (Plk4) in human cells induces centrosome amplification through the simultaneous generation of multiple procentrioles adjoining each parental centriole. This provided an opportunity for dissecting centriole assembly and characterizing assembly intermediates. Critical components were identified and ordered into an assembly pathway through siRNA and localized through immunoelectron microscopy. Plk4, hSas-6, CPAP, Cep135, gamma-tubulin, and CP110 were required at different stages of procentriole formation and in association with different centriolar structures. Remarkably, hSas-6 associated only transiently with nascent procentrioles, whereas Cep135 and CPAP formed a core structure within the proximal lumen of both parental and nascent centrioles. Finally, CP110 was recruited early and then associated with the growing distal tips, indicating that centrioles elongate through insertion of alpha-/beta-tubulin underneath a CP110 cap. Collectively, these data afford a comprehensive view of the assembly pathway underlying centriole biogenesis in human cells.

The human Polo-like kinase 1 (PLK1) and its functional homologues that are present in other eukaryotes have multiple, crucial roles in meiotic and mitotic cell division. By contrast, the functions of other mammalian Polo family members remain largely unknown. Plk4 is the most structurally divergent Polo family member; it is maximally expressed in actively dividing tissues and is essential for mouse embryonic development. Here, we identify Plk4 as a key regulator of centriole duplication. Both gain- and loss-of-function experiments demonstrate that Plk4 is required--in cooperation with Cdk2, CP110 and Hs-SAS6--for the precise reproduction of centrosomes during the cell cycle. These findings provide an attractive explanation for the crucial function of Plk4 in cell proliferation and have implications for the role of Polo kinases in tumorigenesis.

After duplication of the centriole pair during S phase, the centrosome functions as a single microtubule-organizing center until the onset of mitosis, when the duplicated centrosomes separate for bipolar spindle formation. The mechanisms regulating centrosome cohesion and separation during the cell cycle are not well understood. In this study, we analyze the protein rootletin as a candidate centrosome linker component. As shown by immunoelectron microscopy, endogenous rootletin forms striking fibers emanating from the proximal ends of centrioles. Moreover, rootletin interacts with C-Nap1, a protein previously implicated in centrosome cohesion. Similar to C-Nap1, rootletin is phosphorylated by Nek2 kinase and is displaced from centrosomes at the onset of mitosis. Whereas the overexpression of rootletin results in the formation of extensive fibers, small interfering RNA-mediated depletion of either rootletin or C-Nap1 causes centrosome splitting, suggesting that both proteins contribute to maintaining centrosome cohesion. The ability of rootletin to form centriole-associated fibers suggests a dynamic model for centrosome cohesion based on entangling filaments rather than continuous polymeric linkers.