The Human Polo-like Kinase 4 is a Regulator of Centrosome Duplication

Polo-like kinase 4 (Plk4), also known as Snk/Fnk-akin kinase (Sak), is the most structurally divergent member of the Polo-like kinase family (Fode et al., 1994; Leung et al., 2002) and has been associated with cell proliferation (Fode et al., 1994). Most importantly, Plk4-/- mice were found to be embryonically lethal whereas heterozygous Plk+/- mice exhibited an increased incidence of tumorigenesis (Hudson et al., 2001; Ko et al., 2005). However, very little data has been reported on the function of human Plk4. Here, we embarked on a functional characterisation of human Plk4 in cultured cells. This kinase localised to the centrosome throughout the cell cycle, and overexpression studies revealed that an excess of wild type Plk4 induced the overduplication of centrioles in cells. This process was dependent on the cyclin-dependent kinase (Cdk) 2 in addition to CP110 and human Sas-6, two proteins shown to be involved in centriole duplication (Chen et al., 2002b; Leidel et al., 2005). Conversely, depletion of endogenous Plk4 resulted in an inhibition of centriole duplication leading to a loss of centrioles from dividing cells. These data from gain-of-function and loss-of-function experiments demonstrated that Plk4 is an essential regulator of centriole duplication. Further microscopic studies revealed that excess Plk4 activity resulted in the formation of rosette-like structures around centrioles. These structures recruited CPAP which is the human homologue of Sas-4, a protein required for centriole duplication in the nematode worm (Caenorhabditis elegans). We propose that these structures represent centriole precursors. The study presented here is a starting point for further work. The identification of Plk4 as a central regulator of centriole duplication will allow the characterisation of upstream and downstream regulatory pathways, whereas a continued search for interaction partners should resolve the function of this kinase. In particular, the analysis of rosette-like centriole precursors is a promising avenue to provide insights into the mechanism of centriole duplication

A Complex of Two Centrosomal Proteins, CAP350 and FOP, Cooperates with EB1 in Microtubule Anchoring

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, p150(Glued), 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

Plk4-induced centriole biogenesis in human cells

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 Polo kinase Plk4 functions in centriole duplication

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