Rho-dependent control of anillin behavior during cytokinesis

Anillin is a conserved protein required for cytokinesis but its molecular function is unclear. Anillin accumulation at the cleavage furrow is Rho guanine nucleotide exchange factor (GEF)Pbl–dependent but may also be mediated by known anillin interactions with F-actin and myosin II, which are under RhoGEFPbl-dependent control themselves. Microscopy of Drosophila melanogaster S2 cells reveal here that although myosin II and F-actin do contribute, equatorial anillin localization persists in their absence. Using latrunculin A, the inhibitor of F-actin assembly, we uncovered a separate RhoGEFPbl-dependent pathway that, at the normal time of furrowing, allows stable filamentous structures containing anillin, Rho1, and septins to form directly at the equatorial plasma membrane. These structures associate with microtubule (MT) ends and can still form after MT depolymerization, although they are delocalized under such conditions. Thus, a novel RhoGEFPbl-dependent input promotes the simultaneous association of anillin with the plasma membrane, septins, and MTs, independently of F-actin. We propose that such interactions occur dynamically and transiently to promote furrow stability

Rux is a cyclin-dependent kinase inhibitor (CKI) specific for mitotic cyclin–Cdk complexes

AbstractBackground: Roughex (Rux) is a cell-cycle regulator that contributes to the establishment and maintenance of the G1 state in the fruit fly Drosophila. Genetic data show that Rux inhibits the S-phase function of the cyclin A (CycA)–cyclin-dependent kinase 1 (Cdk1) complex; in addition, it can prevent the mitotic functions of CycA and CycB when overexpressed. Rux has no homology to known Cdk inhibitors (CKIs), and the molecular mechanism of Rux function is not known.Results: Rux interacted with CycA and CycB in coprecipitation experiments. Expression of Rux caused nuclear translocation of CycA and CycB, and inhibited Cdk1 but not Cdk2 kinase activity. Cdk1 inhibition by Rux did not rely on inhibitory phosphorylation, disruption of cyclin–Cdk complex formation or changes in subcellular localization. Rux inhibited Cdk1 kinase in two ways: Rux prevented the activating phosphorylation on Cdk1 and also inhibited activated Cdk1 complexes. Surprisingly, Rux had a stimulating effect on CycA–Cdk1 activity when present in low concentrations.Conclusions: Rux fulfils all the criteria for a CKI. This is the first description in a multicellular organism of a CKI that specifically inhibits mitotic cyclin–Cdk complexes. This function of Rux is required for the G1 state and male meiosis and could also be involved in mitotic regulation, while the stimulating effect of Rux might assist in any S-phase function of CycA–Cdk1

Mitotic Regulators Govern Progress through Steps in the Centrosome Duplication Cycle

Centrosome duplication is marked by discrete changes in centriole structure that occur in lockstep with cell cycle transitions. We show that mitotic regulators govern steps in centriole replication in Drosophila embryos. Cdc25string, the expression of which initiates mitosis, is required for completion of daughter centriole assembly. Cdc20fizzy, which is required for the metaphase-anaphase transition, is required for timely disengagement of mother and daughter centrioles. Stabilization of mitotic cyclins, which prevents exit from mitosis, blocks assembly of new daughter centrioles. Common regulation of the nuclear and centrosome cycles by mitotic regulators may ensure precise duplication of the centrosome

Selections that isolate recombinant mitochondrial genomes in animals.

Homologous recombination is widespread and catalyzes evolution. Nonetheless, its existence in animal mitochondrial DNA is questioned. We designed selections for recombination between co-resident mitochondrial genomes in various heteroplasmic Drosophila lines. In four experimental settings, recombinant genomes became the sole or dominant genome in the progeny. Thus, selection uncovers occurrence of homologous recombination in Drosophila mtDNA and documents its functional benefit. Double-strand breaks enhanced recombination in the germline and revealed somatic recombination. When the recombination partner was a diverged Drosophila melanogaster genome or a genome from a different species such as Drosophila yakuba, sequencing revealed long continuous stretches of exchange. In addition, the distribution of sequence polymorphisms in recombinants allowed us to map a selected trait to a particular region in the Drosophila mitochondrial genome. Thus, recombination can be harnessed to dissect function and evolution of mitochondrial genome

Involvement of an SCF(Slmb) complex in timely elimination of E2F upon initiation of DNA replication in Drosophila

BACKGROUND: Cul1 is a core component of the evolutionarily conserved SCF-type ubiquitin ligases that target specific proteins for destruction. SCF action contributes to cell cycle progression but few of the key targets of its action have been identified. RESULTS: We found that expression of the mouse Cul1 (mCul1) in the larval wing disc has a dominant negative effect. It reduces, but does not eliminate, the function of SCF complexes, promotes accumulation of Cubitus interruptus (a target of SCF action), triggers apoptosis, and causes a small wing phenotype. A screen for mutations that dominantly modify this phenotype showed effective suppression upon reduction of E2F function, suggesting that compromised downregulation of E2F contributes to the phenotype. Partial inactivation of Cul1 delayed the abrupt loss of E2F immunofluorescence beyond its normal point of downregulation at the onset of S phase. Additional screens showed that mild reduction in function of the F-box encoding gene slimb enhanced the mCul1 overexpression phenotype. Cell cycle modulation of E2F levels is virtually absent in slimb mutant cells in which slimb function is severely reduced. This implicates Slimb, a known targeting subunit of SCF, in E2F downregulation. In addition, Slimb and E2F interacted in vitro in a phosphorylation-dependent manner. CONCLUSION: We have used genetic and physical interactions to identify the G1/S transcription factor E2F as an SCF(Slmb )target in Drosophila. These results argue that the SCF(Slmb )ubiquitin ligase directs E2F destruction in S phase

A Genome-wide Screen Reveals that Reducing Mitochondrial DNA Polymerase Can Promote Elimination of Deleterious Mitochondrial Mutations.

A mutant mitochondrial genome arising amid the pool of mitochondrial genomes within a cell must compete with existing genomes to survive to the next generation. Even weak selective forces can bias transmission of one genome over another to affect the inheritance of mitochondrial diseases and guide the evolution of mitochondrial DNA (mtDNA). Studies in several systems suggested that purifying selection in the female germline reduces transmission of detrimental mitochondrial mutations [1-7]. In contrast, some selfish genomes can take over despite a cost to host fitness [8-13]. Within individuals, the outcome of competition is therefore influenced by multiple selective forces. The nuclear genome, which encodes most proteins within mitochondria, and all external regulators of mitochondrial biogenesis and dynamics can influence the competition between mitochondrial genomes [14-18], yet little is known about how this works. Previously, we established a Drosophila line transmitting two mitochondrial genomes in a stable ratio enforced by purifying selection benefiting one genome and a selfish advantage favoring the other [8]. Here, to find nuclear genes that impact mtDNA competition, we screened heterozygous deletions tiling ∼70% of the euchromatic regions and examined their influence on this ratio. This genome-wide screen detected many nuclear modifiers of this ratio and identified one as the catalytic subunit of mtDNA polymerase gene (POLG), tam. A reduced dose of tam drove elimination of defective mitochondrial genomes. This study suggests that our approach will uncover targets for interventions that would block propagation of pathogenic mitochondrial mutations

Identification of Drosophila Gene Products Required for Phagocytosis of Candida albicans

Phagocytosis is a highly conserved aspect of innate immunity. We used Drosophila melanogaster S2 cells as a model system to study the phagocytosis of Candida albicans, the major fungal pathogen of humans, by screening an RNAi library representing 7,216 fly genes conserved among metazoans. After rescreening the initial genes identified and eliminating certain classes of housekeeping genes, we identified 184 genes required for efficient phagocytosis of C. albicans. Diverse biological processes are represented, with actin cytoskeleton regulation, vesicle transport, signaling, and transcriptional regulation being prominent. Secondary screens using Escherichia coli and latex beads revealed several genes specific for C. albicans phagocytosis. Characterization of one of those gene products, Macroglobulin complement related (Mcr), shows that it is secreted, that it binds specifically to the surface of C. albicans, and that it promotes its subsequent phagocytosis. Mcr is closely related to the four Drosophila thioester proteins (Teps), and we show that TepII is required for efficient phagocytosis of E. coli (but not C. albicans or Staphylococcus aureus) and that TepIII is required for the efficient phagocytosis of S. aureus (but not C. albicans or E. coli). Thus, this family of fly proteins distinguishes different pathogens for subsequent phagocytosis

Influence of cyclin type and dose on mitotic entry and progression in the early Drosophila embryo

Cyclins are key cell cycle regulators, yet few analyses test their role in timing the events that they regulate. We used RNA interference and real-time visualization in embryos to define the events regulated by each of the three mitotic cyclins of Drosophila melanogaster, CycA, CycB, and CycB3. Each individual and pairwise knockdown results in distinct mitotic phenotypes. For example, mitosis without metaphase occurs upon knockdown of CycA and CycB. To separate the role of cyclin levels from the influences of cyclin type, we knocked down two cyclins and reduced the gene dose of the one remaining cyclin. This reduction did not prolong interphase but instead interrupted mitotic progression. Mitotic prophase chromosomes formed, centrosomes divided, and nuclei exited mitosis without executing later events. This prompt but curtailed mitosis shows that accumulation of cyclin function does not directly time mitotic entry in these early embryonic cycles and that cyclin function can be sufficient for some mitotic events although inadequate for others

Quiescence: early evolutionary origins and universality do not imply uniformity

Cell cycle investigations have focused on relentless exponential proliferation of cells, an unsustainable situation in nature. Proliferation of cells, whether microbial or metazoan, is interrupted by periods of quiescence. The vast majority of cells in an adult metazoan lie quiescent. As disruptions in this quiescence are at the foundation of cancer, it will be important for the field to turn its attention to the mechanisms regulating quiescence. While often presented as a single topic, there are multiple forms of quiescence each with complex inputs, some of which are tied to conceptually challenging aspects of metazoan regulation such as size control. In an effort to expose the enormity of the challenge, I describe the differing biological purposes of quiescence, and the coupling of quiescence in metazoans to growth and to the structuring of tissues during development. I emphasize studies in the organism rather than in tissue culture, because these expose the diversity of regulation. While quiescence is likely to be a primitive biological process, it appears that in adapting quiescence to its many distinct biological settings, evolution has diversified it. Consideration of quiescence in different models gives us an overview of this diversity