Mass isolation of staged Drosophila pupal intestines for analysis of protein ubiquitylation.

Large quantities of developmentally synchronized pupal intestines are required for biochemistry experiments. Here, we present a protocol for the mass isolation of staged pupal intestines during Drosophila melanogaster development based on buoyancy in sucrose for biochemical evaluation of protein ubiquitylation. We describe steps for crossing flies, preparation of samples, immunoprecipitation of proteins from staged isolated tissues, and analysis of samples by western blot.

PINK1, Keap1, and Rtnl1 regulate selective clearance of endoplasmic reticulum during development.

Selective clearance of organelles, including endoplasmic reticulum (ER) and mitochondria, by autophagy plays an important role in cell health. Here, we describe a developmentally programmed selective ER clearance by autophagy. We show that Parkinson's disease-associated PINK1, as well as Atl, Rtnl1, and Trp1 receptors, regulate ER clearance by autophagy.

Drosophila E93 promotes adult development and suppresses larval responses to ecdysone during metamorphosis.

Pulses of the steroid hormone ecdysone act through transcriptional cascades to direct the major developmental transitions during the Drosophila life cycle. These include the prepupal ecdysone pulse, which occurs 10 ​hours after pupariation and triggers the onset of adult morphogenesis and larval tissue destruction. E93 encodes a transcription factor that is specifically induced by the prepupal pulse of ecdysone, supporting a model proposed by earlier work that it specifies the onset of adult development.

Histological assessment of developmental cell death in pupae.

This protocol describes the embedding and processing of pupae in paraffin to monitor tissue changes during development. Although multiple methods are available to evaluate developmental changes in embryos, imaging detailed changes during metamorphosis is challenging as the animal is enclosed in the cuticle, rendering it inaccessible to whole mount imaging. Here, we present a protocol that focuses on developmental clearance of the larval salivary glands in pupae that can be extended to examine other tissues/stages for similar purposes.

A conserved myotubularin-related phosphatase regulates autophagy by maintaining autophagic flux.

Macroautophagy (autophagy) targets cytoplasmic cargoes to the lysosome for degradation. Like all vesicle trafficking, autophagy relies on phosphoinositide identity, concentration, and localization to execute multiple steps in this catabolic process. Here, we screen for phosphoinositide phosphatases that influence autophagy in Drosophila and identify CG3530.

The NF-κB Factor Relish Regulates Atg1 Expression and Controls Autophagy.

Macroautophagy and cell death both contribute to innate immunity, but little is known about how these processes integrate. Drosophila larval salivary glands require autophagy for developmentally programmed cell death, and innate immune signaling factors increase in these dying cells. Here, we show that the nuclear factor κB (NF-κB) factor Relish, a component of the immune deficiency (Imd) pathway, is required for salivary gland degradation.

The indispensable contribution of s38 protein to ovarian-eggshell morphogenesis in Drosophila melanogaster.

Drosophila chorion represents a remarkable model system for the in vivo study of complex extracellular-matrix architectures. For its organization and structure, s38 protein is considered as a component of major importance, since it is synthesized and secreted during early choriogenesis. However, there is no evidence that proves its essential, or redundant, role in chorion biogenesis.

The Proton-Coupled Monocarboxylate Transporter Hermes Is Necessary for Autophagy during Cell Death.

Nutrient availability influences the production and degradation of materials that are required for cell growth and survival. Autophagy is a nutrient-regulated process that is used to degrade cytoplasmic materials and has been associated with human diseases. Solute transporters influence nutrient availability and sensing, yet we know little about how transporters influence autophagy.

Data of sperm-entry inability in ovarian follicles that are depleted of s36 chorionic protein.

This paper presents data associated with the research article entitled "Targeted downregulation of s36 protein unearths its cardinal role in chorion biogenesis and architecture during oogenesis" [1]. chorion is produced by epithelial follicle cells and one of its functional serving role is egg fertilization through the micropyle, a specialized narrow channel at the anterior tip of the egg [2]. Sperm entry during fertilization is necessary for the egg to complete meiosis [3].

Targeted Downregulation of s36 Protein Unearths its Cardinal Role in Chorion Biogenesis and Architecture during Drosophila melanogaster Oogenesis.

Drosophila chorion represents a model biological system for the in vivo study of gene activity, epithelial development, extracellular-matrix assembly and morphogenetic-patterning control. It is produced during the late stages of oogenesis by epithelial follicle cells and develops into a highly organized multi-layered structure that exhibits regional specialization and radial complexity. Among the six major proteins involved in chorion's formation, the s36 and s38 ones are synthesized first and regulated in a cell type-specific and developmental stage-dependent manner.

Ral GTPase and the exocyst regulate autophagy in a tissue-specific manner.

Autophagy traffics cellular components to the lysosome for degradation. Ral GTPase and the exocyst have been implicated in the regulation of stress-induced autophagy, but it is unclear whether they are global regulators of this process. Here, we investigate Ral function in different cellular contexts in Drosophila and find that it is required for autophagy during developmentally regulated cell death in salivary glands, but does not affect starvation-induced autophagy in the fat body.

Global Proteomic Profiling of Drosophila Ovary: A High-resolution, Unbiased, Accurate and Multifaceted Analysis.

Background: Drosophila melanogaster ovary serves as an attractive model system for the investigation of the cell cycle, death, signaling, migration, differentiation, development and stemness. By employing the 3750/+ heterozygote fly strain that carries specific functions in the follicle cell compartment, and a reliable control in GAL4/UAS-based transgenic technology, we herein characterized the protein-expression profiling of D. melanogaster ovary by applying high-resolution proteomic tools and bioinformatics programs.

Proteasome, but not autophagy, disruption results in severe eye and wing dysmorphia: a subunit- and regulator-dependent process in Drosophila.

Proteasome-dependent and autophagy-mediated degradation of eukaryotic cellular proteins represent the two major proteostatic mechanisms that are critically implicated in a number of signaling pathways and cellular processes. Deregulation of functions engaged in protein elimination frequently leads to development of morbid states and diseases. In this context, and through the utilization of GAL4/UAS genetic tool, we herein examined the in vivo contribution of proteasome and autophagy systems in Drosophila eye and wing morphogenesis.

Detrimental effects of proteasome inhibition activity in Drosophila melanogaster: implication of ER stress, autophagy, and apoptosis.

In eukaryotes, the ubiquitin-proteasome machinery regulates a number of fundamental cellular processes through accurate and tightly controlled protein degradation pathways. We have, herein, examined the effects of proteasome functional disruption in Dmp53 (+/+) (wild-type) and Dmp53 (-/-) Drosophila melanogaster fly strains through utilization of Bortezomib, a proteasome-specific inhibitor. We report that proteasome inhibition drastically shortens fly life-span and impairs climbing performance, while it also causes larval lethality and activates developmentally irregular cell death programs during oogenesis.

Programmed cell death of the ovarian nurse cells during oogenesis of the ladybird beetle Adalia bipunctata (Coleoptera: Coccinellidae).

Programmed cell death (PCD) is an evolutionary conserved and genetically regulated form of cell death, in which the cell plays an active role in its own demise. It is widely recognized that PCD can be morphologically classified into three major types: type I, known as apoptosis, type II, called autophagy, and type III, specified as cytoplasmic cell death. So far, PCD has been morphologically analyzed in certain model insect species of the meroistic polytrophic ovary-type, but has never been examined before in insects carrying meroistic telotrophic ovaries.

Proteasome inhibition induces developmentally deregulated programs of apoptotic and autophagic cell death during Drosophila melanogaster oogenesis.

Ubiquitin/proteasome-mediated degradation of eukaryotic proteins is critically implicated in a number of signalling pathways and cellular processes. To specifically impair proteasome activities, in vitro developing Drosophila melanogaster egg chambers were exposed to the MG132 or epoxomicin proteasome inhibitors, while a GAL4/UAS binary genetic system was employed to generate double transgenic flies overexpressing β2 and β6 conditional mutant proteasome subunits in a cell type-specific manner. MG132 and epoxomicin administration resulted in severe deregulation of in vitro developing egg chambers, which was tightly associated with precocious induction of nurse cell-specific apoptotic and autophagic death programmes, featured by actin cytoskeleton disorganization, nuclear chromatin condensation, DRICE caspase activation and autophagosome accumulation.