Centrioles are found in the centrosome core and, as basal bodies, at the base of cilia and flagella. Centriole assembly and duplication is controlled by Polo-like-kinase 4 (Plk4): these processes fail if Plk4 is downregulated and are promoted by Plk4 overexpression. Here we show that the centriolar protein Asterless (Asl; human orthologue CEP152) provides a conserved molecular platform, the amino terminus of which interacts with the cryptic Polo box of Plk4 whereas the carboxy terminus interacts with the centriolar protein Sas-4 (CPAP in humans). Drosophila Asl and human CEP152 are required for the centrosomal loading of Plk4 in Drosophila and CPAP in human cells, respectively. Depletion of Asl or CEP152 caused failure of centrosome duplication; their overexpression led to de novo centriole formation in Drosophila eggs, duplication of free centrosomes in Drosophila embryos, and centrosome amplification in cultured Drosophila and human cells. Overexpression of a Plk4-binding-deficient mutant of Asl prevented centriole duplication in cultured cells and embryos. However, this mutant protein was able to promote microtubule organizing centre (MTOC) formation in both embryos and oocytes. Such MTOCs had pericentriolar material and the centriolar protein Sas-4, but no centrioles at their core. Formation of such acentriolar MTOCs could be phenocopied by overexpression of Sas-4 in oocytes or embryos. Our findings identify independent functions for Asl as a scaffold for Plk4 and Sas-4 that facilitates self-assembly and duplication of the centriole and organization of pericentriolar material.

SummaryCentrioles are 9-fold symmetrical structures at the core of centrosomes and base of cilia whose dysfunction has been linked to a wide range of inherited diseases and cancer [1]. Their duplication is regulated by a protein kinase of conserved structure, the C. elegans ZYG-1 or its Polo-like kinase 4 (Plk4) counterpart in other organisms [2–4]. Although Plk4’s centriolar partners and mechanisms that regulate its stability are known, its crucial substrates for centriole duplication have never been identified. Here we show that Drosophila Plk4 phosphorylates four conserved serines in the STAN motif of the core centriole protein Ana2 to enable it to bind and recruit its Sas6 partner. Ana2 and Sas6 normally load onto both mother and daughter centrioles immediately after their disengagement toward the end of mitosis to seed procentriole formation. Nonphosphorylatable Ana2 still localizes to the centriole but can no longer recruit Sas6 and centriole duplication fails. Thus, following centriole disengagement, recruitment of Ana2 and its phosphorylation by Plk4 are the earliest known events in centriole duplication to recruit Sas6 and thereby establish the architecture of the new procentriole engaged with its parent.

The RNA binding protein Larp1 was originally shown to be involved in spermatogenesis, embryogenesis and cell-cycle progression in Drosophila. Our data show that mammalian Larp1 is found in a complex with poly A binding protein and eukaryote initiation factor 4E and is associated with 60S and 80S ribosomal subunits. A reduction in Larp1 expression by siRNA inhibits global protein synthesis rates and results in mitotic arrest and delayed cell migration. Consistent with these data we show that Larp1 protein is present at the leading edge of migrating cells and interacts directly with cytoskeletal components. Taken together, these data suggest a role for Larp1 in facilitating the synthesis of proteins required for cellular remodelling and migration.

The conserved process of centriole duplication requires Plk4 kinase to recruit and promote interactions between Sas6 and Sas5/Ana2/STIL (respective nomenclature of worms/flies/humans). Plk4-mediated phosphorylation of Ana2/STIL in its conserved STAN motif has been shown to promote its interaction with Sas6. However, STAN motif phosphorylation is not required for recruitment of Ana2 to the centriole. Here we show that in Drosophila, Ana2 loads onto the site of procentriole formation ahead of Sas6 in a process that also requires Plk4. However, whereas Plk4 is first recruited to multiple sites around the ring of zone II at the periphery of the centriole, Ana2 is recruited to a single site in telophase before Plk4 becomes finally restricted to this same single site. When we over-ride the auto-destruction of Plk4, it remains localized to multiple sites in the outer ring of the centriole and, if catalytically active, recruits Ana2 to these sites. Thus, it is the active form of Plk4 that promotes Ana2's recruitment to the centriole. We now show that Plk4 phosphorylates Ana2 at a site other than the STAN motif, which lies in a conserved region we term the ANST (ANa2-STil) motif. Mutation of this site, S38, to a non-phosphorylatable residue prevents the procentriole loading of Ana2 and blocks centriole duplication. Thus the initiation of procentriole formation requires Plk4 to first phosphorylate a single serine residue in the ANST motif to promote Ana2's recruitment and, secondly, to phosphorylate four residues in the STAN motif enabling Ana2 to recruit Sas6. We discuss these findings in light of the multiple Plk4 phosphorylation sites on Ana2.

Myosins constitute a superfamily of motor proteins that convert energy from ATP hydrolysis into mechanical movement along the actin filaments. Phylogenetic analysis currently places myosins into 17 classes based on class-specific features of their conserved motor domain. Traditionally, the myosins have been divided into two classes depending on whether they form monomers or dimers. The conventional myosin of muscle and nonmuscle cells forms class II myosins. They are complex molecules of four light chains bound to two heavy chains that form bipolar filaments via interactions between their coiled-coil tails (type II). Class I myosins are smaller monomeric myosins referred to as unconventional myosins. Now, at least 15 other classes of unconventional myosins are known. How many myosins are needed to ensure the proper development and function of eukaryotic organisms? Thus far, three types of myosins were found in budding yeast, six in the nematode Caenorhabditis elegans, and at least 12 in human. Here, we report on the identification and classification of Drosophila melanogaster myosins. Analysis of the Drosophila genome sequence identified 13 myosin genes. Phylogenetic analysis based on the sequence comparison of the myosin motor domains, as well as the presence of the class-specific domains, suggests that Drosophila myosins can be divided into nine major classes. Myosins belonging to previously described classes I, II, III, V, VI, and VII are present. Molecular and phylogenetic analysis indicates that the fruitfly genome contains at least five new myosins. Three of them fall into previously described myosin classes I, VII, and XV. Another myosin is a homolog of the mouse and human PDZ-containing myosins, forming the recently defined class XVIII myosins. PDZ domains are named after the postsynaptic density, disc-large, ZO-1 proteins in which they were first described. The fifth myosin shows a unique domain composition and a low homology to any of the existing classes. We propose that this is classified when similar myosins are identified in other species.

We demonstrate that a Drosophila Golgi protein, Gorab, is present not only in the trans-Golgi but also in the centriole cartwheel where, complexed to Sas6, it is required for centriole duplication. In addition to centriole defects, flies lacking Gorab are uncoordinated due to defects in sensory cilia, which lose their 9-fold symmetry. We demonstrate the separation of centriole and Golgi functions of Drosophila Gorab in two ways: First, we have created Gorab variants that are unable to localize to trans-Golgi but can still rescue the centriole and cilia defects of gorab null flies. Second, we show that expression of C-terminally tagged Gorab disrupts Golgi functions in cytokinesis of male meiosis, a dominant phenotype overcome by mutations preventing Golgi targeting. Our findings suggest that during metazoan evolution, a Golgi protein has arisen with a second, apparently independent, role in centriole duplication.

The Broad-Complex (BR-C) is an early ecdysone response gene that functions during metamorphosis and encodes a family of zinc-finger transcription factors. It is expressed in a dynamic pattern during oogenesis. Its late expression in the lateral-dorsal-anterior follicle cells is related to the morphogenesis of the chorionic appendages. All four zinc-finger isoforms are expressed in oogenesis, which is consistent with the abnormal appendage phenotypes resulting from their ectopic expression. We investigated the mechanism by which the BR-C affects chorion deposition by using BrdU to follow the effects of BR-C misexpression on DNA replication and in situ hybridization to ovarian mRNA to evaluate chorion gene expression. Ectopic BR-C expression leads to prolonged endoreplication and to additional amplification of genes, besides the chorion genes, at other sites in the genome. The pattern of chorion gene expression is not affected along the anterior-posterior axis, but the follicle cells at the anterior of the oocyte fail to migrate correctly in an anterior direction when BR-C is misexpressed. We conclude that the target genes of the BR-C in oogenesis include a protein essential for endoreplication and chorion gene amplification. This may provide a link between steroid hormones and the control of DNA replication during oogenesis.

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