Centrosome amplification can initiate tumorigenesis in flies.

Centrosome amplification is a common feature of many cancer cells, and it has been previously proposed that centrosome amplification can drive genetic instability and so tumorigenesis. To test this hypothesis, we generated Drosophila lines that have extra centrosomes in approximately 60% of their somatic cells. Many cells with extra centrosomes initially form multipolar spindles, but these spindles ultimately become bipolar. This requires a delay in mitosis that is mediated by the spindle assembly checkpoint (SAC). As a result of this delay, there is no dramatic increase in genetic instability in flies with extra centrosomes, and these flies maintain a stable diploid genome over many generations. The asymmetric division of the larval neural stem cells, however, is compromised in the presence of extra centrosomes, and larval brain cells with extra centrosomes can generate metastatic tumors when transplanted into the abdomens of wild-type hosts. Thus, centrosome amplification can initiate tumorigenesis in flies.

Protein Phosphatase 1 Down Regulates ZYG-1 Levels to Limit Centriole Duplication.

In humans perturbations of centriole number are associated with tumorigenesis and microcephaly, therefore appropriate regulation of centriole duplication is critical. The C. elegans homolog of Plk4, ZYG-1, is required for centriole duplication, but our understanding of how ZYG-1 levels are regulated remains incomplete. We have identified the two PP1 orthologs, GSP-1 and GSP-2, and their regulators I-2SZY-2 and SDS-22 as key regulators of ZYG-1 protein levels. We find that down-regulation of PP1 activity either directly, or by mutation of szy-2 or sds-22 can rescue the loss of centriole duplication associated with a zyg-1 hypomorphic allele. Suppression is achieved through an increase in ZYG-1 levels, and our data indicate that PP1 normally regulates ZYG-1 through a post-translational mechanism. While moderate inhibition of PP1 activity can restore centriole duplication to a zyg-1 mutant, strong inhibition of PP1 in a wild-type background leads to centriole amplification via the production of more than one daughter centriole. Our results thus define a new pathway that limits the number of daughter centrioles produced each cycle.

The C. elegans F-box proteins LIN-23 and SEL-10 antagonize centrosome duplication by regulating ZYG-1 levels.

The correct segregation of DNA during cell division requires formation of a bipolar spindle, organized at each pole by a centrosome. The regulation of centrosome duplication such that each mitotic cell has exactly two centrosomes is therefore of central importance to cell division. Deregulation of centrosome duplication causes the appearance of supernumerary centrosomes, which are a hallmark of many cancer cells and can contribute to tumorigenesis. Overexpression of the kinase Plk4, which is required for centrosome duplication, causes the formation of extra centrosomes, and aberrant Plk4 expression levels are associated with cancer. Data from Drosophila and human cells show that Plk4 levels are regulated by the SCF ubiquitin ligase and proteasomal degradation. Recognition of Plk4 by the SCF complex is mediated by the F-box protein Slimb/ßTrCP. We show that levels of the C. elegans Plk4 homolog ZYG-1 are elevated by impairing proteasome or SCF function, indicating that ZYG-1 is regulated by a conserved mechanism. In C. elegans, similar to Drosophila and humans, we find that the Slimb/ßTrCP homolog LIN-23 regulates ZYG-1 levels. In addition, we show that a second F-box protein, SEL-10, also contributes to ZYG-1 regulation. Co-depletion of LIN-23 and SEL-10 suggests these proteins function cooperatively. Because SEL-10 is the homolog of human FBW7, which is frequently mutated in cancer, our findings have implications for understanding tumorigenesis.

MAP9/MAPH-9 supports axonemal microtubule doublets and modulates motor movement.

Microtubule doublets (MTDs) comprise an incomplete microtubule (B-tubule) attached to the side of a complete cylindrical microtubule. These compound microtubules are conserved in cilia across the tree of life; however, the mechanisms by which MTDs form and are maintained in vivo remain poorly understood. Here, we identify microtubule-associated protein 9 (MAP9) as an MTD-associated protein. We demonstrate that C. elegans MAPH-9, a MAP9 homolog, is present during MTD assembly and localizes exclusively to MTDs, a preference that is in part mediated by tubulin polyglutamylation. We find that loss of MAPH-9 causes ultrastructural MTD defects, including shortened and/or squashed B-tubules with reduced numbers of protofilaments, dysregulated axonemal motor velocity, and perturbed cilia function. Because we find that the mammalian ortholog MAP9 localizes to axonemes in cultured mammalian cells and mouse tissues, we propose that MAP9/MAPH-9 plays a conserved role in regulating ciliary motors and supporting the structure of axonemal MTDs.

The C. elegans deglutamylase CCPP-6 does not operate redundantly with CCPP-1 in gross cilia integrity.

Tubulin glutamylation is a reversible modification of the microtubules that regulates cilia stability and function. The addition of glutamates to the microtubule is catalyzed by members of the TTLL family of enzymes, while the removal is carried out by a family of cytosolic carboxypeptidase (CCP) enzymes. C. elegans has two deglutamylating enzymes, CCPP-1 and CCPP-6 . CCPP-1 is required for ciliary stability and function in the worm, however CCPP-6 is dispensable for cilia integrity. To investigate redundancy between the two deglutamylating enzymes we made a ccpp-1 ( ok1821 ); ccpp-6 ( ok382 ) double mutant. The double mutant shows normal viability, and the dye-filling phenotypes are not worse than the ccpp-1 single mutant, suggesting that CCPP-1 and CCPP-6 do not function redundantly in C. elegans cilia .

A ccpp-6 deletion mutation does not impair gross cilia integrity in C. elegans.

Tubulin glutamylation is a reversible modification that regulates microtubule function in cilia. The removal of glutamylation from microtubules is carried out by a family of cytosolic carboxypeptidase (CCP) enzymes. C. elegans has two deglutamylating enzymes, CCPP-1 and CCPP-6, homologs of mammalian CCP1 and CCP5 respectively. CCPP-1 is required for ciliary stability and function. To determine whether CCPP-6 is similarly required for cilia integrity in C. elegans we analyzed the ccpp-6(ok382) deletion mutant. We find that both dye-filling and male mating are normal, suggesting that CCPP-6 is not required for ciliary integrity in C. elegans.

Overexpressing centriole-replication proteins in vivo induces centriole overduplication and de novo formation.

BACKGROUND: Centrosomes have important roles in many aspects of cell organization, and aberrations in their number and function are associated with various diseases, including cancer. Centrosomes consist of a pair of centrioles surrounded by a pericentriolar matrix (PCM), and their replication is tightly regulated. Here, we investigate the effects of overexpressing the three proteins known to be required for centriole replication in Drosophila-DSas-6, DSas-4, and Sak. RESULTS: By directly observing centriole replication in living Drosophila embryos, we show that the overexpression of GFP-DSas-6 can drive extra rounds of centriole replication within a single cell cycle. Extra centriole-like structures also accumulate in brain cells that overexpress either GFP-DSas-6 or GFP-Sak, but not DSas-4-GFP. No extra centrioles accumulate in spermatocytes that overexpress any of these three proteins. Most remarkably, the overexpression of any one of these three proteins results in the rapid de novo formation of many hundreds of centriole-like structures in unfertilized eggs, which normally do not contain centrioles. CONCLUSIONS: Our data suggest that the levels of centriolar DSas-6 determine the number of daughter centrioles formed during centriole replication. Overexpression of either DSas-6 or Sak can induce the formation of extra centrioles in some tissues but not others, suggesting that centriole replication is regulated differently in different tissues. The finding that the overexpression of DSas-4, DSas-6, or Sak can rapidly induce the de novo formation of centriole-like structures in Drosophila eggs suggests that this process results from the stabilization of centriole-precursors that are normally present in the egg.

Caenorhabditis elegans glutamylating enzymes function redundantly in male mating.

Microtubule glutamylation is an important modulator of microtubule function and has been implicated in the regulation of centriole stability, neuronal outgrowth and cilia motility. Glutamylation of the microtubules is catalyzed by a family of tubulin tyrosine ligase-like (TTLL) enzymes. Analysis of individual TTLL enzymes has led to an understanding of their specific functions, but how activities of the TTLL enzymes are coordinated to spatially and temporally regulate glutamylation remains relatively unexplored. We have undertaken an analysis of the glutamylating TTLL enzymes in C. elegans We find that although all five TTLL enzymes are expressed in the embryo and adult worm, loss of individual enzymes does not perturb microtubule function in embryonic cell divisions. Moreover, normal dye-filling, osmotic avoidance and male mating behavior indicate the presence of functional amphid cilia and male-specific neurons. A ttll-4(tm3310); ttll-11(tm4059); ttll-5(tm3360) triple mutant, however, shows reduced male mating efficiency due to a defect in the response step, suggesting that these three enzymes function redundantly, and that glutamylation is required for proper function of the male-specific neurons.

The Drosophila Pericentrin-like-protein (PLP) cooperates with Cnn to maintain the integrity of the outer PCM.

Centrosomes comprise a pair of centrioles surrounded by a matrix of pericentriolar material (PCM). In vertebrate cells, Pericentrin plays an important part in mitotic PCM assembly, but the Drosophila Pericentrin-like protein (PLP) appears to have a more minor role in mitotic fly cells. Here we investigate the function of PLP during the rapid mitotic cycles of the early Drosophila embryo. Unexpectedly, we find that PLP is specifically enriched in the outer-most regions of the PCM, where it largely co-localizes with the PCM scaffold protein Cnn. In the absence of PLP the outer PCM appears to be structurally weakened, and it rapidly disperses along the centrosomal microtubules (MTs). As a result, centrosomal MTs are subtly disorganized in embryos lacking PLP, although mitosis is largely unperturbed and these embryos develop and hatch at near-normal rates. Y2H analysis reveals that PLP can potentially form multiple interactions with itself and with the PCM recruiting proteins Asl, Spd-2 and Cnn. A deletion analysis suggests that PLP participates in a complex network of interactions that ultimately help to strengthen the PCM.

Everything in moderation: Proteolytic regulation of centrosome duplication.

The presence of too many or too few centrosomes at mitosis can disrupt the timely formation of a bipolar spindle and may lead to aneuploidy and cancer. Strict control of centrosome duplication is therefore crucial. Centrosome duplication must occur once per cell cycle and the number of new centrioles made must be tightly controlled. The importance of protein degradation for the orderly progression of the cell cycle has long been recognized, but until recently the role of proteolysis in the regulation of centrosome duplication had not been appreciated. Recent evidence suggests that restricting protein levels so that a single new centriole is built next to each pre-existing centriole is one way in which centrosome duplication is controlled. Here we discuss our recent finding that the SCF ubiquitin ligase complex regulates centrosome duplication in C. elegans in the larger context of the proteolytic regulation of centrosome duplication.

CGEF-1 and CHIN-1 regulate CDC-42 activity during asymmetric division in the Caenorhabditis elegans embryo.

The anterior-posterior axis of the Caenorhabditis elegans embryo is elaborated at the one-cell stage by the polarization of the partitioning (PAR) proteins at the cell cortex. Polarization is established under the control of the Rho GTPase RHO-1 and is maintained by the Rho GTPase CDC-42. To understand more clearly the role of the Rho family GTPases in polarization and division of the early embryo, we constructed a fluorescent biosensor to determine the localization of CDC-42 activity in the living embryo. A genetic screen using this biosensor identified one positive (putative guanine nucleotide exchange factor [GEF]) and one negative (putative GTPase activating protein [GAP]) regulator of CDC-42 activity: CGEF-1 and CHIN-1. CGEF-1 was required for robust activation, whereas CHIN-1 restricted the spatial extent of CDC-42 activity. Genetic studies placed CHIN-1 in a novel regulatory loop, parallel to loop described previously, that maintains cortical PAR polarity. We found that polarized distributions of the nonmuscle myosin NMY-2 at the cell cortex are independently produced by the actions of RHO-1, and its effector kinase LET-502, during establishment phase and CDC-42, and its effector kinase MRCK-1, during maintenance phase. CHIN-1 restricted NMY-2 recruitment to the anterior during maintenance phase, consistent with its role in polarizing CDC-42 activity during this phase.

Centrioles: some self-assembly required.

Centrioles play an important role in organizing microtubules and are precisely duplicated once per cell cycle. New (daughter) centrioles typically arise in association with existing (mother) centrioles (canonical assembly), suggesting that mother centrioles direct the formation of daughter centrioles. However, under certain circumstances, centrioles can also selfassemble free of an existing centriole (de novo assembly). Recent work indicates that the canonical and de novo pathways utilize a common mechanism and that a mother centriole spatially constrains the self-assembly process to occur within its immediate vicinity. Other recently identified mechanisms further regulate canonical assembly so that during each cell cycle, one and only one daughter centriole is assembled per mother centriole.

A small family of sushi-class retrotransposon-derived genes in mammals and their relation to genomic imprinting.

Ty3/gypsy retrotransposons are rare in mammalian genomes despite their abundance in invertebrate and other vertebrate classes. Here we identify a family of nine conserved mammalian genes with homology to Ty3/gypsy retrotransposons but which have lost their ability to autonomously retrotranspose. Of these, five map to the X chromosome while the remaining four are autosomal. Comparative phylogenetic analyses show them to have strongest homology to the sushi-ichi element from Fugu rubripes. Two of the autosomal gene members, Peg10 and Rtl1, are known to be imprinted, being expressed from the paternally inherited chromosome homologue. This suggests, consistent with the host-parasite response theory of the evolution of the imprinting mechanism, that parental-origin specific epigenetic control may be mediated by genomic "parasitic" elements such as these. Alternatively, these elements may preferentially integrate into regions that are differentially modified on the two homologous chromosomes such as imprinted domains and the X chromosome and acquire monoallelic expression. We assess the imprinting status of the remaining autosomal members of this family and show them to be biallelically expressed in embryo and placenta. Furthermore, the methylation status of Rtl1 was assayed throughout development and was found to resemble that of actively, silenced repetitive elements rather than imprinted sequences. This indicates that the ability to undergo genomic imprinting is not an inherent property of all members of this family of retroelements. Nonetheless, the conservation but functional divergence between the different members suggests that they have undergone positive selection and acquired distinct endogenous functions within their mammalian hosts.