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.

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 anchoring of microtubules (MTs) to subcellular structures is critical for cell shape, polarity, and motility. In mammalian cells, the centrosome is a prominent MT anchoring structure. A number of proteins, including ninein, p150Glued , and EB1, have been implicated in centrosomal MT anchoring, but the process is far from understood. Here we show that CAP350 and FOP (FGFR1 oncogene partner) form a centrosomal complex required for MT anchoring. We show that the C-terminal domain of CAP350 interacts directly with FOP and that both proteins localize to the centrosome throughout the cell cycle. FOP also binds to EB1 and is required for localizing EB1 to the centrosome. Depletion of either CAP350, FOP, or EB1 by siRNA causes loss of MT anchoring and profound disorganization of the MT network. These results have implications for the mechanisms underlying MT anchoring at the centrosome and they attribute a key MT anchoring function to two novel centrosomal proteins, CAP350 and FOP. INTRODUCTIONIn most animal cells, the centrosome plays an important role in the organization of MT networks (Rieder et al., 2001;Bornens, 2002;Nigg, 2004;Ou and Rattner, 2004;Doxsey et al., 2005). A single centrosome is composed of two centrioles that are surrounded by amorphous pericentriolar material (PCM). These two centrioles (sometimes referred to as mother and daughter) differ in structure, function and age (state of maturity). In particular, the fully mature centriole is characterized by the presence of appendages at its distal end. MTs are nucleated from so-called ␥-tubulin ring complexes (␥-TuRCs; Moritz et al., 2004). These ring-shaped multiprotein complexes are present within PCM associated with both mother and daughter centrioles and, indeed, both centrioles are competent to nucleate MTs . Subsequent to nucleation, MTs are released (Keating et al., 1997;Abal et al., 2002). So, in order to remain associated with centrosomes, they need to be captured by centrosomal MT anchoring activities (Dammermann et al., 2003). Anchoring mechanisms remain incompletely understood, but the available evidence suggests that appendages of the mature centriole play a prominent role in MT anchoring . Moreover, transport of released MTs to appendages was shown to require dynein/dynactin activity Clark and Meyer, 1999;Quintyne et al., 1999).Several proteins have been shown to localize to centriole appendages. These include ninein (Mogensen et al., 2000), odf2/cenexin (Lange and Gull, 1995;Nakagawa et al., 2001;Ishikawa et al., 2005), centriolin (Gromley et al., 2003), ⑀-tubulin (Chang et al., 2003), Cep170 (Guarguaglini et al., 2005), and CEP110 (Ou et al., 2002). An involvement in MT anchoring has been clearly demonstrated for ninein (Mogensen et al., 2000;Dammermann and Merdes, 2002;Abal et al., 2002;Delgehyr et al., 2005). However, not all proteins implicated in MT anchoring are concentrated at appendages. In particular, evidence for a role in MT anchoring has been reported for PCM-1 (Dammermann and Merdes, 2002), BBS4 (Kim et al., 2004), and CEP1...