Ribosomopathy-associated mutations cause proteotoxic stress that is alleviated by TOR inhibition.

Ribosomes are multicomponent molecular machines that synthesize all of the proteins of living cells. Most of the genes that encode the protein components of ribosomes are therefore essential. A reduction in gene dosage is often viable albeit deleterious and is associated with human syndromes, which are collectively known as ribosomopathies1-3. The cell biological basis of these pathologies has remained unclear. Here, we model human ribosomopathies in Drosophila and find widespread apoptosis and cellular stress in the resulting animals. This is not caused by insufficient protein synthesis, as reasonably expected. Instead, ribosomal protein deficiency elicits proteotoxic stress, which we suggest is caused by the accumulation of misfolded proteins that overwhelm the protein degradation machinery. We find that dampening the integrated stress response4 or autophagy increases the harm inflicted by ribosomal protein deficiency, suggesting that these activities could be cytoprotective. Inhibition of TOR activity-which decreases ribosomal protein production, slows down protein synthesis and stimulates autophagy5-reduces proteotoxic stress in our ribosomopathy model. Interventions that stimulate autophagy, combined with means of boosting protein quality control, could form the basis of a therapeutic strategy for this class of diseases.

Chromatin dynamics in regeneration epithelia: Lessons from Drosophila imaginal discs.

During the process of regeneration, a switch in the transcription program occurs in cells that contribute to the reconstruction of the missing tissue. Early signals released upon damage are integrated into the chromatin of responding cells to change its activity and function. Changes in chromatin dynamics result in transcriptional reprogramming, this is the coordinated regulation of expression of a specific subset of genes required for the regeneration process. Here we summarize changes in gene expression and chromatin dynamics that occurs during the process of regeneration of Drosophila imaginal discs.

Accuracy of the First Stage of Hepatotoxicity Testing with the Use of Drosophila melanogaster Test System.

The genotoxic effects of heterocyclic compounds were evaluated on the basis of genetic and toxicological characteristics of a biological model of Drosophila melanogaster. Analysis of the viability parameters (fertility, progeny mortality) showed that of 6 tested substance, substance No. 3 exhibited minimum toxicity. After application of substances No. 1 and No. 5 in the studied concentrations, the number of survived flies was insufficient for further analysis, which attested to high toxicity of these substances. The intensity of apoptosis was studied in response to substances Nos. 2, 4, and 6. Substance No. 4 proved to be optimal by the parameter toxicity/apoptosis (low toxicity/high apoptosis), while substance No. 3 exhibited low toxicity, which manifested in low apoptosis intensity.

Trans-generational transmission of altered phenotype resulting from flubendiamide-induced changes in apoptosis in larval imaginal discs of Drosophila melanogaster.

The eye and wing morphology of Drosophila melanogaster maintain unique, stable pattern of genesis from larval eye and wing imaginal discs. Increased apoptosis in cells of eye and wing discs was found to be associated with flubendiamide (fluoride containing insecticide) exposure (at the range 0.25-10µg/mL) in D. melanogaster larvae. The chemical fed larvae on attaining adulthood revealed alterations in morphology and symmetry of their compound eyes and wings through scanning electron microscopy. Nearly 40% and 30% of flies (P generation) demonstrated alterations in eyes and wings respectively. Transmission electron microscopic study (at the range 1-20µg/mL) also established variation in the rhabdomere and pigment cell orientation as well as in the shape of the ommatidium. Subsequent SEM study with F1 and F2 generation flies also revealed structural variation in eye and wing. Decrease in percentage of altered eye and wing phenotype was noted in subsequent generations (P> F1>F2). Thus, the diamide insecticide, flubendiamide, expected to be environmentally safe at sub-lethal concentrations was found to increase apoptosis in larvae and thereby cause morphological alteration in the adult D. melanogaster. This study further demonstrated trans-generational transmission of altered phenotype in three subsequent generations of a non-target insect model, D. melanogaster.

Cell dynamics underlying oriented growth of the Drosophila wing imaginal disc.

Quantitative analysis of the dynamic cellular mechanisms shaping the Drosophila wing during its larval growth phase has been limited, impeding our ability to understand how morphogen patterns regulate tissue shape. Such analysis requires explants to be imaged under conditions that maintain both growth and patterning, as well as methods to quantify how much cellular behaviors change tissue shape. Here, we demonstrate a key requirement for the steroid hormone 20-hydroxyecdysone (20E) in the maintenance of numerous patterning systems in vivo and in explant culture. We find that low concentrations of 20E support prolonged proliferation in explanted wing discs in the absence of insulin, incidentally providing novel insight into the hormonal regulation of imaginal growth. We use 20E-containing media to observe growth directly and to apply recently developed methods for quantitatively decomposing tissue shape changes into cellular contributions. We discover that whereas cell divisions drive tissue expansion along one axis, their contribution to expansion along the orthogonal axis is cancelled by cell rearrangements and cell shape changes. This finding raises the possibility that anisotropic mechanical constraints contribute to growth orientation in the wing disc.

Rapid Detection of γ-H2Av Foci in Ex Vivo MMS-Treated Drosophila Imaginal Discs.

In Drosophila melanogaster, DNA double-strand breaks (DSBs) created by exposure to gamma or X-ray radiation can be quantified by immunofluorescent detection of phosphorylated histone H2Av (γ-H2Av) foci in imaginal disc tissues. This technique has been less useful for studying DSBs in imaginal discs exposed to DSB-inducing chemicals, since standard protocols require raising larvae in food treated with liquid chemical suspensions. These protocols typically take 3-4 days to complete and result in heterogeneous responses that do not provide information about the kinetics of DSB formation and repair. Here, we describe a novel and rapid method to quantify DSBs in imaginal discs cultured ex vivo with methyl methanesulfonate (MMS) or other DSB-inducing chemicals. The described method requires less than 24 h and provides precise control over MMS concentration and exposure time, enabling reproducible detection of transient DSBs. Furthermore, this technique can be used for nearly any chemical treatment and can be modified and adapted for several different experimental setups and downstream molecular analyses.

Cultivation and Live Imaging of Drosophila Imaginal Discs.

The ex vivo cultivation and live imaging of wing discs open exciting new research avenues by overcoming the limitations of end-point analysis of fixed tissues. Here we describe how to prepare an optimized wing disc culture medium (WM1) and how to dissect and arrange wing discs for cultivation and live imaging. This protocol enables the study of dynamic phenomena such as cell division and delamination as well as the use of pharmacological compounds and biosensors. Wing discs cultured and imaged as described here, maintain constant levels of proliferation during the first ten hours of culture.

Systematic functional characterization of putative zinc transport genes and identification of zinc toxicosis phenotypes in Drosophila melanogaster.

The heavy metal zinc is an essential component of the human diet and is incorporated as a structural component in up to 10% of all mammalian proteins. The physiological importance of zinc homeostasis at the cellular level and the molecular mechanisms involved in this process have become topics of increasing interest in recent years. We have performed a systematic functional characterization of the majority of the predicted Drosophila Zip (zinc/iron regulated transporter-related protein) and ZnT genes, using the Gal4-UAS system to carry out both ubiquitous and targeted over-expression and suppression studies for 13 of the 17 putative zinc transport genes identified to date. We found that six of these 13 genes may be essential for fly viability and that three of the remaining seven demonstrate over-expression phenotypes. Our findings reaffirm the previously proposed function of dZnT63C (CG17723: FBgn005432) as an important zinc efflux protein and indicate that the fly homolog of hZip1, dZip42C.1 (CG9428: FBgn0033096), is a strong zinc importer in Drosophila. By combining over-expression of dZip42C.1 with suppression of dZnT63C we were able to produce easily identifiable zinc toxicosis phenotypes, which can be rescued or worsened by modifying dietary zinc content. Our findings show that a genetically based zinc toxicosis situation can be therapeutically treated or exacerbated by modifications to the diet, providing a sensitized background for future, more detailed studies of Zip/ZnT function.

[Nuclear texture in mitotic cells].

Changes of nuclear texture in mitotic cells of Drosophila melanogaster imaginal discs were studied. The distribution of voxels DAPI fluorescence intensities was used as the quantitative measure of the nuclear texture. The integral characteristics such as the portion of voxels with a given fluorescent signal level and autocorrelation of pixel intensities were used. We showed the nuclear texture has specific changes at different mitotic stages and this can be used for more precise staging of mitosis. Colchicines treatment pathologies, connected to abnormal mitoses, by nuclear-texture approach.