Protocol for assessing translation in living Drosophila imaginal discs by O-propargyl-puromycin incorporation.

Translation is a fundamental process of cellular behavior. Here, we present a protocol for measuring translation in Drosophila epithelial tissues using O-propargyl-puromycin (OPP), a puromycin derivative. We detail steps for larval dissection, OPP incorporation, fixation, OPP labeling, immunostaining, and imaging.

Ribosomal protein mutations and cell competition: autonomous and nonautonomous effects on a stress response.

Ribosomal proteins (Rps) are essential for viability. Genetic mutations affecting Rp genes were first discovered in Drosophila, where they represent a major class of haploinsufficient mutations. One mutant copy gives rise to the dominant "Minute" phenotype, characterized by slow growth and small, thin bristles.

The transcription factor Xrp1 orchestrates both reduced translation and cell competition upon defective ribosome assembly or function.

Ribosomal Protein () gene haploinsufficiency affects translation rate, can lead to protein aggregation, and causes cell elimination by competition with wild type cells in mosaic tissues. We find that the modest changes in ribosomal subunit levels observed were insufficient for these effects, which all depended on the AT-hook, bZip domain protein Xrp1. Xrp1 reduced global translation through PERK-dependent phosphorylation of eIF2α.

Cell competition removes segmental aneuploid cells from imaginal disc-derived tissues based on ribosomal protein gene dose.

Aneuploidy causes birth defects and miscarriages, occurs in nearly all cancers and is a hallmark of aging. Individual aneuploid cells can be eliminated from developing tissues by unknown mechanisms. Cells with ribosomal protein () gene mutations are also eliminated, by cell competition with normal cells.

Drosophila RpS12 controls translation, growth, and cell competition through Xrp1.

Whereas complete loss of Rp function is generally lethal, most heterozygous Rp mutants grow more slowly and are subject to competitive loss from mosaics tissues that also contain wild type cells. The rpS12 gene has a special role in the cell competition of other Ribosomal Protein (Rp) mutant cells in Drosophila. Elimination by cell competition is promoted by higher RpS12 levels and prevented by a specific rpS12 mis-sense mutation, identifying RpS12 as a key effector of cell competition due to mutations in other Rp genes.

A potential link between p53, cell competition and ribosomopathy in mammals and in Drosophila.

The term cell competition has been used to describe the phenomenon whereby particular cells can be eliminated during tissue growth only when more competitive cells are available to replace them. Multiple examples implicate differential activity of p53 in cell competition in mammals, but p53 has not been found to have the same role in Drosophila, where the phenomenon of cell competition was first recognized. Recent studies now show that Drosophila cells harboring mutations in Ribosomal protein (Rp) genes, which are eliminated by cell competition with wild type cells, activate a p53 target gene, Xrp1.

A Regulatory Response to Ribosomal Protein Mutations Controls Translation, Growth, and Cell Competition.

Ribosomes perform protein synthesis but are also involved in signaling processes, the full extent of which are still being uncovered. We report that phenotypes of mutating ribosomal proteins (Rps) are largely due to signaling. Using Drosophila, we discovered that a bZip-domain protein, Xrp1, becomes elevated in Rp mutant cells.

Ribosomal Protein S12e Has a Distinct Function in Cell Competition.

Wild-type Drosophila cells can remove cells heterozygous for ribosomal protein mutations (known as "Minute" mutant cells) from genetic mosaics, a process termed cell competition. The ribosomal protein S12 was unusual because cells heterozygous for rpS12 mutations were not competed by wild-type, and a viable missense mutation in rpS12 protected Minute cells from cell competition with wild-type cells. Furthermore, cells with Minute mutations were induced to compete with one another by altering the gene dose of rpS12, eliminating cells with more rpS12 than their neighbors.

bHLH proteins involved in Drosophila neurogenesis are mutually regulated at the level of stability.

Proneural bHLH activators are expressed in all neuroectodermal regions prefiguring events of central and peripheral neurogenesis. Drosophila Sc is a prototypical proneural activator that heterodimerizes with the E-protein Daughterless (Da) and is antagonized by, among others, the E(spl) repressors. We determined parameters that regulate Sc stability in Drosophila S2 cells.

The two Tribolium E(spl) genes show evolutionarily conserved expression and function during embryonic neurogenesis.

Tribolium castaneum is a well-characterised model insect, whose short germ-band mode of embryonic development is characteristic of many insect species and differs from the exhaustively studied Drosophila. Mechanisms of early neurogenesis, however, show significant conservation with Drosophila, as a characteristic pattern of neuroblasts arises from neuroectoderm proneural clusters in response to the bHLH activator Ash, a homologue of Achaete-Scute. Here we study the expression and function of two other bHLH proteins, the bHLH-O repressors E(spl)1 and E(spl)3.

Essential roles of Da transactivation domains in neurogenesis and in E(spl)-mediated repression.

E proteins are a special class of basic helix-loop-helix (bHLH) proteins that heterodimerize with many bHLH activators to regulate developmental decisions, such as myogenesis and neurogenesis. Daughterless (Da) is the sole E protein in Drosophila and is ubiquitously expressed. We have characterized two transcription activation domains (TADs) in Da, called activation domain 1 (AD1) and loop-helix (LH), and have evaluated their roles in promoting peripheral neurogenesis.