A Wnt/beta-catenin pathway antagonist Chibby binds Cenexin at the distal end of mother centrioles and functions in primary cilia formation.

The mother centriole of the centrosome is distinguished from immature daughter centrioles by the presence of accessory structures (distal and subdistal appendages), which play an important role in the organization of the primary cilium in quiescent cells. Primary cilia serve as sensory organelles, thus have been implicated in mediating intracellular signal transduction pathways. Here we report that Chibby (Cby), a highly conserved antagonist of the Wnt/ß-catenin pathway, is a centriolar component specifically located at the distal end of the mother centriole and essential for assembly of the primary cilium. Cby appeared as a discrete dot in the middle of a ring-like structure revealed by staining with a distal appendage component of Cep164. Cby interacted with one of the appendage components, Cenexin (Cnx), which thereby abrogated the inhibitory effect of Cby on ß-catenin-mediated transcriptional activation in a dose-dependent manner. Cby and Cnx did not precisely align, as Cby was detected at a more distal position than Cnx. Cnx emerged earlier than Cby during the cell cycle and was required for recruitment of Cby to the mother centriole. However, Cby was dispensable for Cnx localization to the centriole. During massive centriogenesis in in vitro cultured mouse tracheal epithelial cells, Cby and Cnx were expressed in a similar pattern, which was coincident with the expression of Foxj1. Our results suggest that Cby plays an important role in organization of both primary and motile cilia in collaboration with Cnx.

Centrosome amplification in CHO and DT40 cells by inactivation of cyclin-dependent kinases.

To study the mechanism of centrosome duplication in cycling cells, we established a novel system of multiple centrosome formation in two types of cells: CHO cells treated with RO3306, a Cyclin-dependent kinase 1 (Cdk1) inhibitor and DT40 cells, in which Cdks were knocked out by chemical genetics. Cdk1-inactivated cells initiated DNA replication and centrosome duplication at the onset of S phase. They became arrested at the end of G2, but the centrosome cycle continued to produce supernumerary centrioles/centrosomes without DNA endoreplication in those cells. Centrosomes were amplified in a highly synchronous and reproducible manner: all of them were located next to the nucleus and spread widely apart from each other with several µm in distance. Double knockout of Cdk1 and Cdk2 caused cell cycle arrest at G1/S and centrosomes were no longer duplicated. However, cells continued to grow and increased their volume over 10-fold during 48 hr of culture. Centrosome components, including γ-tubulin and Cep135, were synthesized and accumulated during the arrest, allowing rapid centrosome multiplication upon recovery from the cell cycle arrest or expression of exogenous Plk4 in G1/S cells. Thus centrosome amplification results from the discoordination of the centrosome cycle from the progression of other cell cycle events, which is controlled by different levels of Cdk activities.

Functional screen of human MCM2-7 variant alleles for disease-causing potential.

Origin licensing builds a fundamental basis for genome stability in DNA replication. Recent studies reported that deregulation of origin licensing is associated with replication stress in precancerous lesions. The heterohexameric complex of minichromosome maintenance proteins (MCM2-7 complex) plays an essential role in origin licensing. Previously, we reported the recovery of the first viable Mcm mutant allele (named Mcm4(Chaos3)) in mice. The Mcm4(Chaos3) allele destabilizes the MCM2-7 complex, leading to chromosome instability and the formation of spontaneous tumors in Mcm4(Chaos3) homozygous mice. Supporting our finding, a recent study reported that mice with reduced expression of MCM2 die with lymphomas within the first few months after birth. These data strongly suggest that mutant Mcm2-7 genes are cancer-causing genes with nearly complete penetrance in mice. This could be the case for humans as well. Nevertheless, related investigations have not been undertaken due to the essential nature of the MCM2-7 genes. To circumvent this problem, we focused on the variant alleles of human MCM2-7 genes derived from single nucleotide polymorphisms. We created a total of 14 variant alleles in the corresponding genes in Saccharomyces cerevisiae. The phenotypic consequence was assayed for minichromosome loss, a surrogate phenotype for genome instability and cancer susceptibility. This screen identified a MCM5 variant allele with pathogenic potential. This allele deserves further investigations on its effect on cancer development in human populations.