Andrew J. Simmonds

Gawky is a component of cytoplasmic mRNA processing bodies required for early Drosophila development

In mammalian cells, the GW182 protein localizes to cytoplasmic bodies implicated in the regulation of messenger RNA (mRNA) stability, translation, and the RNA interference pathway. Many of these functions have also been assigned to analogous yeast cytoplasmic mRNA processing bodies. We have characterized the single Drosophila melanogaster homologue of the human GW182 protein family, which we have named Gawky (GW). Drosophila GW localizes to punctate, cytoplasmic foci in an RNA-dependent manner. Drosophila GW bodies (GWBs) appear to function analogously to human GWBs, as human GW182 colocalizes with GW when expressed in Drosophila cells. The RNA-induced silencing complex component Argonaute2 and orthologues of LSm4 and Xrn1 (Pacman) associated with 5′–3′ mRNA degradation localize to some GWBs. Reducing GW activity by mutation or antibody injection during syncytial embryo development leads to abnormal nuclear divisions, demonstrating an early requirement for GWB-mediated cytoplasmic mRNA regulation. This suggests that gw represents a previously unknown member of a small group of genes that need to be expressed zygotically during early embryo development.

RNA Interference Effector Proteins Localize to Mobile Cytoplasmic Puncta in Schizosaccharomyces pombe

Ago1, Dcr1 and Rdp1 are the core components of the RNA interference (RNAi) apparatus in the fission yeast Schizosaccharomyces pombe. They function in distinct gene‐silencing pathways that direct homology‐dependent degradation of mRNA and modification of chromatin. In addition, Ago1 and Dcr1 regulate enactment of Cdc2‐dependent cell cycle checkpoints. The ability of the RNAi apparatus to perform multiple roles in these divergent pathways is sure to require dynamic localization of Ago1, Dcr1 and/or Rdp1. Although limited information is available, comprehensive studies regarding the relative localizations of Ago1, Dcr1 and Rdp1 are lacking. To this end, we employed live‐cell imaging and immunoelectron microscopy to study the intracellular localizations of these proteins. In contrast to previous reports, our study results indicate that the bulk of Ago1 and Dcr1 form stable complexes and are associated with large, mobile, highly dynamic cytoplasmic elements. The majority of Rdp1 is localized to the nucleus, but a pool of Rdp1 is associated with the same cytoplasmic structures. The movements of these structures were dependent upon ATP and intact microtubules. Recruitment of the RNAi core proteins to these structures was not dependent upon siRNAs. Together, our data indicate that the enzymes required for the initiation and effector phases of RNA‐dependent gene silencing are concentrated in a common intracellular location, an arrangement that would be expected to result in highly efficient post‐transcriptional gene silencing.

Functional characterization of the Drosophila MRP (mitochondrial RNA processing) RNA gene

MRP RNA is a noncoding RNA component of RNase mitochondrial RNA processing (MRP), a multi-protein eukaryotic endoribonuclease reported to function in multiple cellular processes, including ribosomal RNA processing, mitochondrial DNA replication, and cell cycle regulation. A recent study predicted a potential Drosophila ortholog of MRP RNA (CR33682) by computer-based genome analysis. We have confirmed the expression of this gene and characterized the phenotype associated with this locus. Flies with mutations that specifically affect MRP RNA show defects in growth and development that begin in the early larval period and end in larval death during the second instar stage. We present several lines of evidence demonstrating a role for Drosophila MRP RNA in rRNA processing. The nuclear fraction of Drosophila MRP RNA localizes to the nucleolus. Further, a mutant strain shows defects in rRNA processing that include a defect in 5.8S rRNA processing, typical of MRP RNA mutants in other species, as well as defects in early stages of rRNA processing.

Peroxisome-Mediated Metabolism Is Required for Immune Response to Microbial Infection

Summary The innate immune response is critical for animal homeostasis and is conserved from invertebrates to vertebrates. This response depends on specialized cells that recognize, internalize, and destroy microbial invaders through phagocytosis. This is coupled to autonomous or non‐autonomous cellular signaling via reactive oxygen species (ROS) and cytokine production. Lipids are known signaling factors in this process, as the acute phase response of macrophages is accompanied by systemic lipid changes that help resolve inflammation. We found that peroxisomes, membrane‐enclosed organelles central to lipid metabolism and ROS turnover, were necessary for the engulfment of bacteria by Drosophila and mouse macrophages. Peroxisomes were also required for resolution of bacterial infection through canonical innate immune signaling. Reduced peroxisome function impaired the turnover of the oxidative burst necessary to fight infection. This impaired response to bacterial challenge affected cell and organism survival and revealed a previously unknown requirement for peroxisomes in phagocytosis and innate immunity. Graphical Abstract Figure. No Caption available. HighlightsPeroxisomes are necessary for phagocytosis by Drosophila and mouse macrophagesPeroxisomes are required for resolution of microbial infectionPeroxisomes modulate innate immune pathways necessary to fight infectionLack of functional peroxisomes affects organism survival in response to infection &NA; Peroxisomes are organelles involved in lipid metabolism and reactive oxygen species turnover. Di Cara et al. now show that peroxisomes are required to resolve microbial infection by innate immunity. Peroxisomes assist in the progression of phagocytosis and activate innate immune signaling to promote survival in the face of microbial challenge.

A Systematic Cell-Based Analysis of Localization of Predicted Drosophila Peroxisomal Proteins

Peroxisomes are membrane‐bound organelles found in almost all eukaryotic cells. They perform specialized biochemical functions that vary with organism, tissue or cell type. Mutations in human genes required for the assembly of peroxisomes result in a spectrum of diseases called the peroxisome biogenesis disorders. A previous sequence‐based comparison of the predicted proteome of Drosophila melanogaster (the fruit fly) to human proteins identified 82 potential homologues of proteins involved in peroxisomal biogenesis, homeostasis or metabolism. However, the subcellular localization of these proteins relative to the peroxisome was not determined. Accordingly, we tested systematically the localization and selected functions of epitope‐tagged proteins in Drosophila Schneider 2 cells to determine the subcellular localization of 82 potential Drosophila peroxisomal protein homologues. Excluding the Pex proteins, 34 proteins localized primarily to the peroxisome, 8 showed dual localization to the peroxisome and other structures, and 26 localized exclusively to organelles other than the peroxisome. Drosophila is a well‐developed laboratory animal often used for discovery of gene pathways, including those linked to human disease. Our work establishes a basic understanding of peroxisome protein localization in Drosophila. This will facilitate use of Drosophila as a genetically tractable, multicellular model system for studying key aspects of human peroxisome disease.

Functional characterization of peroxisome biogenic proteins Pex5 and Pex7 of Drosophila

Peroxisomes are ubiquitous membrane-enclosed organelles involved in lipid processing and reactive oxygen detoxification. Mutations in human peroxisome biogenesis genes (Peroxin, PEX) cause progressive developmental disabilities and, in severe cases, early death. PEX5 and PEX7 are receptors that recognize different peroxisomal targeting signals called PTS1 and PTS2, respectively, and traffic proteins to the peroxisomal matrix. We characterized mutants of Drosophila melanogaster Pex5 and Pex7 and found that adult animals are affected in lipid processing. Moreover, Pex5 mutants exhibited severe developmental defects in the embryonic nervous system and muscle, similar to what is observed in humans with Pex5 mutations, while Pex7 fly mutants were weakly affected in brain development, suggesting different roles for Pex7 in fly and human. Of note, although no PTS2-containing protein has been identified in Drosophila, Pex7 from Drosophila can function as a bona fide PTS2 receptor because it can rescue targeting of the PTS2-containing protein Thiolase to peroxisomes in PEX7 mutant human fibroblasts.

Moesin is involved in polarity maintenance and cortical remodelling during asymmetric cell division

An intact actomyosin network is essential for asymmetric cell division; however, the precise mechanisms remain unclear. We find that the actin-binding protein Moesin is required for neuroblast proliferation and mitotic progression. In addition, the asymmetric and dynamic distribution of Moesin drives the cortical remodeling of dividing neuroblasts, contributing to polarity maintenance and cell size asymmetry.

The activity of the Drosophila Vestigial protein is modified by Scalloped-dependent phosphorylation

The Drosophila vestigial gene is required for proliferation and differentiation of the adult wing and for differentiation of larval and adult muscle identity. Vestigial is part of a multi-protein transcription factor complex, which includes Scalloped, a TEAD-class DNA binding protein. Binding Scalloped is necessary for translocation of Vestigial into the nucleus. We show that Vestigial is extensively post-translationally modified and at least one of these modifications is required for proper function during development. We have shown that there is p38-dependent phosphorylation of Serine 215 in the carboxyl-terminal region of Vestigial. Phosphorylation of Serine 215 occurs in the nucleus and requires the presence of Scalloped. Comparison of a phosphomimetic and non-phosphorylatable mutant forms of Vestigial shows differences in the ability to rescue the wing and muscle phenotypes associated with a null vestigial allele.

Gawky (GW) is the Drosophila melanogaster GW182 Homologue

While the human GW182 gene was discovered over 10 years ago, functional characterization of the Drosophila melanogaster GW182 othologue—Gawky (Gw, previously denoted as CG31992, CG11484, CG9905, or dGW182) has been relatively recent. (Rehwinkel et al. 2005; Schneider et al. 2006) However, the Drosophila model has contributed greatly to studying the role(s) of the GW182 family proteins in multiple pathways and in particular their role in RNA interference (RNAi). Of the commonly used metazoan models, Drosophila is unique in that there is only one Gw protein encoded by the Drosophila genome and this homologue retains a high level of sequence and/or organizational identity to vertebrate GW182 proteins (Fig. 8.1). Thus, the potential functional redundancy associated with the multiple GW182 family proteins encoded by the mammalian genome is less of a concern in Drosophila studies (Schneider et al. 2006; Eystathioy et al. 2002). The bulk of the currently published literature regarding Drosophila Gw can be divided into two main categories. Functional studies describing the Drosophila gw mutant phenotype and cell-biological/biochemical studies probing the vital role of Gw in the mechanics of Drosophila miRNA pathway.

Imaging the cellular dynamics of Drosophila Argonaute proteins.

Drosophila melanogaster is used extensively as a model system to uncover genetic and molecular pathways that regulate various cellular activities. There are five members of the Argonaute protein family in Drosophila. Argonautes have been found to be localized to cytoplasmic ribonucleoprotein containing structures in both cultured Drosophila cells and developing embryos. However, in fixed cell preparations some Drosophila Argonaute family proteins co-localize with structures containing known as RNA processing (P) body components while others do not. The ability to image Argonaute family proteins in live Drosophila cells, (both cultured and within developing embryos) allows for accurate genetic dissection of the pathways involved in the assembly, mobility, disassembly, and other dynamic processes of Argonaute-containing bodies. Here we describe a method of rapidly creating vectors for, and assay the activity of, fluorescently tagged Argonaute proteins in cultured Drosophila cells and embryos.