Preparing Adult Drosophila melanogaster for Whole Brain Imaging during Behavior and Stimuli Responses.

We present a method developed specifically to image the whole Drosophila brain during ongoing behavior such as walking. Head fixation and dissection are optimized to minimize their impact on behavior. This is first achieved by using a holder that minimizes movement hindrances. The back of the fly's head is glued to this holder at an angle that allows optical access to the whole brain while retaining the fly's ability to walk, groom, smell, taste and see. The back of the head is dissected to remove tissues in the optical path and muscles responsible for head movement artefacts. The fly brain can subsequently be imaged to record brain activity, for instance using calcium or voltage indicators, during specific behaviors such as walking or grooming, and in response to different stimuli. Once the challenging dissection, which requires considerable practice, has been mastered, this technique allows to record rich data sets relating whole brain activity to behavior and stimulus responses.

Transient Receptor Potential Canonical (TRPC) Channels: Then and Now.

Twenty-five years ago, the first mammalian Transient Receptor Potential Canonical (TRPC) channel was cloned, opening the vast horizon of the TRPC field. Today, we know that there are seven TRPC channels (TRPC1-7). TRPCs exhibit the highest protein sequence similarity to the Drosophila melanogaster TRP channels. Similar to Drosophila TRPs, TRPCs are localized to the plasma membrane and are activated in a G-protein-coupled receptor-phospholipase C-dependent manner. TRPCs may also be stimulated in a store-operated manner, via receptor tyrosine kinases, or by lysophospholipids, hypoosmotic solutions, and mechanical stimuli. Activated TRPCs allow the influx of Ca2+ and monovalent alkali cations into the cytosol of cells, leading to cell depolarization and rising intracellular Ca2+ concentration. TRPCs are involved in the continually growing number of cell functions. Furthermore, mutations in the TRPC6 gene are associated with hereditary diseases, such as focal segmental glomerulosclerosis. The most important recent breakthrough in TRPC research was the solving of cryo-EM structures of TRPC3, TRPC4, TRPC5, and TRPC6. These structural data shed light on the molecular mechanisms underlying TRPCs' functional properties and propelled the development of new modulators of the channels. This review provides a historical overview of the major advances in the TRPC field focusing on the role of gene knockouts and pharmacological tools.

Circadian Entrainment of Drosophila Melanogaster.

Nearly universal among organisms, circadian rhythms coordinate biological activity to earth's orbit around the sun. To identify factors creating this rhythm and to understand the resulting outputs, entrainment of model organisms to defined circadian time-points is required. Here we detail a procedure to entrain many Drosophila to a defined circadian rhythm. Furthermore, we detail post-entrainment steps to prepare samples for immunofluorescence, nucleic acid, or protein extraction-based analysis.

Sargassum fusiforme Fucoidan SP2 Extends the Lifespan of Drosophila melanogaster by Upregulating the Nrf2-Mediated Antioxidant Signaling Pathway.

Damage accumulated in the genome and macromolecules is largely attributed to increased oxidative damage and a lack of damage repair in a cell, and this can eventually trigger the process of aging. Alleviating the extent of oxidative damage is therefore considered as a potential way to promote longevity. SFPS, a heteropolysaccharide extracted from the brown alga Sargassum fusiforme, has previously been shown to alleviate oxidative damage during the aging process in mice, but whether SFPS could extend the lifespan of an organism was not demonstrated. Furthermore, the precise component within SFPS that is responsible for the antioxidant activity and the underlying mechanism of such activity was also not resolved. In this study, SP2, a fucoidan derived from SFPS, was shown to exhibit strong antioxidant activity as measured by in vitro radical-scavenging assays. SP2 also improved the survival rate of D. melanogaster subjected to oxidative stress. The flies that were fed with a diet containing SP2 from the time of eclosion displayed significant enhancement in lifespan and reduced accumulation of triglyceride at the old-age stage. In addition, SP2 markedly improved the activities of the antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) and reduced the contents of the malondialdehyde (MDA) and oxidized glutathione (GSSG) in old flies. Furthermore, SP2 also upregulated the expression levels of the nuclear factor-erythroid-2-like 2 (nfe2l2 or nrf2) and its downstream target genes, accompanied by a dramatic reduction in the expression of kelch-like ECH-associated protein 1 (keap1, a canonical inhibitor of the Nrf2) in old flies. Additional support linking the crucial role of the Nrf2/ARE pathway to the antioxidant effect of SP2 was the relatively high survival rate under heat stress for D. melanogaster individuals receiving SP2 supplement, an effect that was abolished by the inclusion of inhibitors specific for the Nrf2/ARE pathway. Collectively, the results indicated that SP2, a S. fusiforme fucoidan, could promote longevity in D. melanogaster by enhancing the Nrf2-mediated antioxidant signaling pathway during the aging process.

The CCR4-NOT complex is a tumor suppressor in Drosophila melanogaster eye cancer models.

BACKGROUND: The CNOT3 protein is a subunit of the CCR4-NOT complex, which is involved in mRNA degradation. We recently identified CNOT3 loss-of-function mutations in patients with T-cell acute lymphoblastic leukemia (T-ALL). METHODS: Here, we use different Drosophila melanogaster eye cancer models to study the potential tumor suppressor function of Not3, the CNOT3 orthologue, and other members of the CCR4-NOT complex. RESULTS: Our data show that knockdown of Not3, the structural components Not1/Not2, and the deadenylases twin/Pop2 all result in increased tumor formation. In addition, overexpression of Not3 could reduce tumor formation. Not3 downregulation has a mild but broad effect on gene expression and leads to increased levels of genes involved in DNA replication and ribosome biogenesis. CycB upregulation also contributes to the Not3 tumor phenotype. Similar findings were obtained in human T-ALL cell lines, pointing out the conserved function of Not3. CONCLUSIONS: Together, our data establish a critical role for Not3 and the entire CCR4-NOT complex as tumor suppressor.

The TreadWheel: Interval Training Protocol for Gently Induced Exercise in Drosophila melanogaster.

The incidence of complex metabolic diseases has increased as a result of a widespread transition towards lifestyles of increased caloric intake and lowered activity levels. These multifactorial diseases arise from a combination of genetic, environmental, and behavioral factors. One such complex disease is Metabolic Syndrome (MetS), which is a cluster of metabolic disorders, including hypertension, hyperglycemia, and abdominal obesity. Exercise and dietary intervention are the primary treatments recommended by doctors to mitigate obesity and its subsequent metabolic diseases. Exercise intervention, in particular aerobic interval training, stimulates favorable changes in the common risk factors for Type 2 Diabetes Mellitus (T2DM), Cardiovascular Disease (CVD), and other conditions. With the influx of evidence describing the therapeutic effect exercise has on metabolic health, establishing a system that models exercise in a controlled setting provides a valuable tool for assessing the effects of exercise in an experimental context. Drosophila melanogaster is a great tool for investigating the physiological and molecular changes that result from exercise intervention. The flies have short lifespans and similar mechanisms of metabolizing nutrients when compared to humans. To induce exercise in Drosophila, we developed a machine called the TreadWheel, which utilizes the fly's innate, negative geotaxis tendency to gently induce climbing. This enables researchers to perform experiments on large cohorts of genetically diverse flies to better understand the genotype-by-environment interactions underlying the effects of exercise on metabolic health.

A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System.

The regrowth capacity of damaged neurons governs neuroregeneration and functional recovery after nervous system trauma. Over the past few decades, various intrinsic and extrinsic inhibitory factors involved in the restriction of axon regeneration have been identified. However, simply removing these inhibitory cues is insufficient for successful regeneration, indicating the existence of additional regulatory machinery. Drosophila melanogaster, the fruit fly, shares evolutionarily conserved genes and signaling pathways with vertebrates, including humans. Combining the powerful genetic toolbox of flies with two-photon laser axotomy/dendriotomy, we describe here the Drosophila sensory neuron - dendritic arborization (da) neuron injury model as a platform for systematically screening for novel regeneration regulators. Briefly, this paradigm includes a) the preparation of larvae, b) lesion induction to dendrite(s) or axon(s) using a two-photon laser, c) live confocal imaging post-injury and d) data analysis. Our model enables highly reproducible injury of single labeled neurons, axons, and dendrites of well-defined neuronal subtypes, in both the peripheral and central nervous system.

Modelling Cooperative Tumorigenesis in Drosophila.

The development of human metastatic cancer is a multistep process, involving the acquisition of several genetic mutations, tumour heterogeneity, and interactions with the surrounding microenvironment. Due to the complexity of cancer development in mammals, simpler model organisms, such as the vinegar fly, Drosophila melanogaster, are being utilized to provide novel insights into the molecular mechanisms involved. In this review, we highlight recent advances in modelling tumorigenesis using the Drosophila model, focusing on the cooperation of oncogenes or tumour suppressors, and the interaction of mutant cells with the surrounding tissue in epithelial tumour initiation and progression.

JNK and Yorkie drive tumor progression by generating polyploid giant cells in Drosophila.

Epithelial cancer tissues often possess polyploid giant cells, which are thought to be highly oncogenic. However, the mechanisms by which polyploid giant cells are generated in tumor tissues and how such cells contribute to tumor progression remain elusive. We previously noticed in Drosophila imaginal epithelium that cells mutant for the endocytic gene rab5 exhibit enlarged nuclei. Here we find that mutations in endocytic 'neoplastic tumor-suppressor' genes, such as rab5, vps25, erupted, or avalanche result in generation of polyploid giant cells. Genetic analyses on rab5-defective cells reveal that cooperative activation of JNK and Yorkie generates polyploid giant cells via endoreplication. Mechanistically, Yorkie-mediated upregulation of Diap1 cooperates with JNK to downregulate the G2/M cyclin CycB, thereby inducing endoreplication. Interestingly, malignant tumors induced by Ras activation and cell polarity defect also consist of polyploid giant cells, which are generated by JNK and Yorkie-mediated downregulation of CycB. Strikingly, elimination of polyploid giant cells from such malignant tumors by blocking endoreplication strongly suppressed tumor growth and metastatic behavior. Our observations suggest that JNK and Yorkie, two oncogenic proteins activated in many types of human cancers, cooperatively drive tumor progression by generating oncogenic polyploid giant cells.

Detecting Anastasis In Vivo by CaspaseTracker Biosensor.

Anastasis (Greek for "rising to life") is a recently discovered cell recovery phenomenon whereby dying cells can reverse late-stage cell death processes that are generally assumed to be intrinsically irreversible. Promoting anastasis could in principle rescue or preserve injured cells that are difficult to replace such as cardiomyocytes or neurons, thereby facilitating tissue recovery. Conversely, suppressing anastasis in cancer cells, undergoing apoptosis after anti-cancer therapies, may ensure cancer cell death and reduce the chances of recurrence. However, these studies have been hampered by the lack of tools for tracking the fate of cells that undergo anastasis in live animals. The challenge is to identify the cells that have reversed the cell death process despite their morphologically normal appearance after recovery. To overcome this difficulty, we have developed Drosophila and mammalian CaspaseTracker biosensor systems that can identify and permanently track the anastatic cells in vitro or in vivo. Here, we present in vivo protocols for the generation and use of the CaspaseTracker dual biosensor system to detect and track anastasis in Drosophila melanogaster after transient exposure to cell death stimuli. While conventional biosensors and protocols can label cells actively undergoing apoptotic cell death, the CaspaseTracker biosensor can permanently label cells that have recovered after caspase activation - a hallmark of late-stage apoptosis, and simultaneously identify active apoptotic processes. This biosensor can also track the recovery of the cells that attempted other forms of cell death that directly or indirectly involved caspase activity. Therefore, this protocol enables us to continuously track the fate of these cells and their progeny, facilitating future studies of the biological functions, molecular mechanisms, physiological and pathological consequences, and therapeutic implications of anastasis. We also discuss the appropriate controls to distinguish cells that undergo anastasis from those that display non-apoptotic caspase activity in vivo.

Mitochondrial dysfunction induces dendritic loss via eIF2α phosphorylation.

Mitochondria are key contributors to the etiology of diseases associated with neuromuscular defects or neurodegeneration. How changes in cellular metabolism specifically impact neuronal intracellular processes and cause neuropathological events is still unclear. We here dissect the molecular mechanism by which mitochondrial dysfunction induced by Prel aberrant function mediates selective dendritic loss in Drosophila melanogaster class IV dendritic arborization neurons. Using in vivo ATP imaging, we found that neuronal cellular ATP levels during development are not correlated with the progression of dendritic loss. We searched for mitochondrial stress signaling pathways that induce dendritic loss and found that mitochondrial dysfunction is associated with increased eIF2α phosphorylation, which is sufficient to induce dendritic pathology in class IV arborization neurons. We also observed that eIF2α phosphorylation mediates dendritic loss when mitochondrial dysfunction results from other genetic perturbations. Furthermore, mitochondrial dysfunction induces translation repression in class IV neurons in an eIF2α phosphorylation-dependent manner, suggesting that differential translation attenuation among neuron subtypes is a determinant of preferential vulnerability.

Genetic changes accompanying the evolution of host specialization in Drosophila sechellia.

Changes in host specialization contribute to the diversification of phytophagous insects. When shifting to a new host, insects evolve new physiological, morphological, and behavioral adaptations. Our understanding of the genetic changes responsible for these adaptations is limited. For instance, we do not know how often host shifts involve gain-of-function vs. loss-of-function alleles. Recent work suggests that some genes involved in odor recognition are lost in specialists. Here we show that genes involved in detoxification and metabolism, as well as those affecting olfaction, have reduced gene expression in Drosophila sechellia-a specialist on the fruit of Morinda citrifolia. We screened for genes that differ in expression between D. sechellia and its generalist sister species, D. simulans. We also screened for genes that are differentially expressed in D. sechellia when these flies chose their preferred host vs. when they were forced onto other food. D. sechellia increases expression of genes involved with oogenesis and fatty acid metabolism when on its host. The majority of differentially expressed genes, however, appear downregulated in D. sechellia. For several functionally related genes, this decrease in expression is associated with apparent loss-of-function alleles. For example, the D. sechellia allele of Odorant binding protein 56e (Obp56e) harbors a premature stop codon. We show that knockdown of Obp56e activity significantly reduces the avoidance response of D. melanogaster toward M. citrifolia. We argue that apparent loss-of-function alleles like Obp56e potentially contributed to the initial adaptation of D. sechellia to its host. Our results suggest that a subset of genes reduce or lose function as a consequence of host specialization, which may explain why, in general, specialist insects tend to shift to chemically similar hosts.

Nasal myiasis by fruit fly larvae: a case report.

We report a case of human nasal myiasis caused by fruit fly larvae in a 33-year-old man who visited the south coast of Turkey. The fruit fly (Drosophila melanogaster) is endemic in the southern part of Turkey. Infestation with fruit fly larvae must be considered in patients who are visiting areas of the world where Drosophila is endemic. This is the first reported case of fruit fly larvae causing human nasal myiasis. The clinical presentation and treatment strategies are discussed.