RESUMEN
Ferritins are protein nanocages that accumulate inside their cavity thousands of oxidized iron atoms bound to oxygen and phosphates. Both characteristic types of eukaryotic ferritin subunits are present in secreted ferritins from insects, but here dimers between Ferritin 1 Heavy Chain Homolog (Fer1HCH) and Ferritin 2 Light Chain Homolog (Fer2LCH) are further stabilized by disulfide-bridge in the 24-subunit complex. We addressed ferritin assembly and iron loading in vivo using novel transgenic strains of Drosophila melanogaster. We concentrated on the intestine, where the ferritin induction process can be controlled experimentally by dietary iron manipulation. We showed that the expression pattern of Fer2LCH-Gal4 lines recapitulated iron-dependent endogenous expression of the ferritin subunits and used these lines to drive expression from UAS-mCherry-Fer2LCH transgenes. We found that the Gal4-mediated induction of mCherry-Fer2LCH subunits was too slow to effectively introduce them into newly formed ferritin complexes. Endogenous Fer2LCH and Fer1HCH assembled and stored excess dietary iron, instead. In contrast, when flies were genetically manipulated to co-express Fer2LCH and mCherry-Fer2LCH simultaneously, both subunits were incorporated with Fer1HCH in iron-loaded ferritin complexes. Our study provides fresh evidence that, in insects, ferritin assembly and iron loading in vivo are tightly regulated.
Asunto(s)
Drosophila melanogaster/metabolismo , Enterocitos/metabolismo , Ferritinas/metabolismo , Animales , Animales Modificados Genéticamente , Drosophila melanogaster/genética , Ferritinas/química , Ferritinas/genética , Tracto Gastrointestinal/metabolismo , Expresión Génica , Genes Reporteros , Genotipo , Hierro/metabolismo , Larva , Modelos Biológicos , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Transporte de Proteínas , Proteínas Recombinantes de FusiónRESUMEN
The Drosophila eye is widely used as a model for studies of development and neuronal degeneration. With the powerful mitotic recombination technique, elegant genetic screens based on clonal analysis have led to the identification of signaling pathways involved in eye development and photoreceptor (PR) differentiation at larval stages. We describe here the Tomato/GFP-FLP/FRT method, which can be used for rapid clonal analysis in the eye of living adult Drosophila. Fluorescent photoreceptor cells are imaged with the cornea neutralization technique, on retinas with mosaic clones generated by flipase-mediated recombination. This method has several major advantages over classical histological sectioning of the retina: it can be used for high-throughput screening and has proved an effective method for identifying the factors regulating PR survival and function. It can be used for kinetic analyses of PR degeneration in the same living animal over several weeks, to demonstrate the requirement for specific genes for PR survival or function in the adult fly. This method is also useful for addressing cell autonomy issues in developmental mutants, such as those in which the establishment of planar cell polarity is affected.
Asunto(s)
Drosophila/citología , Proteínas Fluorescentes Verdes/química , Células Fotorreceptoras de Invertebrados/citología , Retina/citología , Animales , Drosophila/genética , Proteínas de Drosophila , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Microscopía Fluorescente/métodos , Células Fotorreceptoras de Invertebrados/química , Células Fotorreceptoras de Invertebrados/metabolismo , Recombinación Genética , Factores de Transcripción/biosíntesis , Factores de Transcripción/química , Factores de Transcripción/genéticaRESUMEN
Metals have vital functions as prosthetic groups in enzymes, but in labile form they can propagate oxidative stress. The primary function of ferritin is to store bioavailable iron in the form of ferrihydrite. In animals, ferritin is also used to traffic and recycle iron, and to modulate intestinal iron absorption. However, the effect of ferritin accumulation on cellular iron bioavailability remains poorly understood. Moreover, putative in vivo interactions of ferritin with other metal ions have been proposed, but their physiological relevance remains unclear. Here, heterozygous mutant and overexpression ferritin strains of Drosophila melanogaster were subjected to dietary iron manipulations to study the dynamics of iron partition between ferritin and other proteins. Quantitative magnetic analysis of whole fly samples indicated that iron loading of the ferritin core varied in the different genotypes. Total paramagnetic iron content, a likely correlate of bioavailable iron, was reduced in flies overexpressing ferritin when compared with control white flies. Further, three-dimensional maps of the ferritin protein shell and iron core were obtained from single particle transmission electron microscopy imaging and confirmed the similarity between Drosophila and Trichoplusia ferritin structures. Purified Drosophila ferritin also contained small amounts of zinc and manganese. Flies that overexpressed ferritin accumulated in their bodies half the amount of manganese compared to their respective controls. Our results indicate that ferritin may be involved in the homeostasis of other divalent metals, besides iron, and that overexpression of ferritin, sometimes employed to rescue neurodegenerative models of disease, serves to limit divalent metal bio-availability in cells.
Asunto(s)
Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Ferritinas/química , Ferritinas/genética , Hierro/química , Animales , Cationes Bivalentes/química , Genotipo , Heterocigoto , Homeostasis , Imagen por Resonancia Magnética , Manganeso/química , Microscopía Electrónica de Transmisión , Mutación , Estrés Oxidativo , Temperatura , Zinc/químicaRESUMEN
We report a new two-color fluorescent imaging system to visualize the mosaic adult photoreceptor neurons (PRs) in real-time. Using this method, we examined a collection of 434 mutants and identified genes required for PR survival, planar cell polarity (PCP), patterning and differentiation. We could track the progression of PR degeneration in living flies. By introducing the expression of p35, a caspase inhibitor, we found mutations that specifically activate caspase-dependent death. Moreover, we showed that grh is required in R3 for correct PCP establishment. The "Tomato/GFP-FLP/FRT" method allows high-throughput, rapid and precise identification of survival and developmental pathways in living adult PRs at single-cell resolution.
Asunto(s)
Apoptosis , Drosophila/fisiología , Células Fotorreceptoras de Invertebrados/fisiología , Animales , Polaridad Celular , Supervivencia Celular , Proteínas de Unión al ADN/fisiología , Proteínas de Drosophila/fisiología , Fluorescencia , Mutación , Factores de Transcripción/fisiologíaRESUMEN
The unfolded protein response (UPR) is a specific cellular process that allows the cell to cope with the overload of unfolded/misfolded proteins in the endoplasmic reticulum (ER). ER stress is commonly associated with degenerative pathologies, but its role in disease progression is still a matter for debate. Here, we found that mutations in the ER-resident chaperone, neither inactivation nor afterpotential A (NinaA), lead to mild ER stress, protecting photoreceptor neurons from various death stimuli in adult Drosophila. In addition, Drosophila S2 cultured cells, when pre-exposed to mild ER stress, are protected from H(2)O(2), cycloheximide- or ultraviolet-induced cell death. We show that a specific ER-mediated signal promotes antioxidant defences and inhibits caspase-dependent cell death. We propose that an immediate consequence of the UPR not only limits the accumulation of misfolded proteins but also protects tissues from harmful exogenous stresses.