EAST and Chromator control the destruction and remodeling of muscles during Drosophila metamorphosis

Metamorphosis involves the destruction of larval, the formation of adult and the transformation of larval into adult tissues. In this study, we demonstrate the role of the Drosophila nuclear proteins EAST and Chromator in tissue destruction and remodeling. To better understand the function of east, we performed a yeast two-hybrid screen and identified the euchromatin associated protein Chromator as a candidate interactor. To analyze the functional significance of our two-hybrid data, we generated a set of novel pupal lethal Chro alleles by P-element excision. The pupal lethal Chro mutants resemble lethal east alleles as homozygous mutants develop into pharates with normal looking body parts, but fail to eclose. The eclosion defect of the Chro alleles is rescued in an east heterozygous background, indicating antagonistic genetic interactions between the two genes. Live cell imaging was applied to study muscle development during metamorphosis. Consistent with the eclosion defects, mutant pharates of both genes show loss and abnormal differentiation of adult eclosion muscles. The two genes have opposite effects on the destruction of larval muscles in metamorphosis. While Chro mutants show incomplete histolysis, muscles degenerate prematurely in east mutants. Moreover east mutants affect the remodeling of abdominal larval muscles into adult eclosion muscles. During this process, loss of east interferes with the spatial coordination of thinning of the larval muscles. Overexpression of EAST-GFP can prevent the disintegration of polytene chromosomes during programmed cell death. We propose that Chro activates and east inhibits processes and genes involved in tissue destruction and remodeling.     

Inter-Ring Communication Is Dispensable in the Reaction Cycle of Group II Chaperonins

Chaperonins are ubiquitous molecular chaperones with the subunit molecular mass of 60 kDa. They exist as double-ring oligomers with central cavities. An ATP-dependent conformational change of the cavity induces the folding of an unfolded protein that is captured in the cavity. In the group I chaperonins, which are present in eubacteria and eukaryotic organelles, inter-ring communication takes important role for the reaction cycle. However, there has been limited study on the inter-ring communication in the group II chaperonins that exist in archaea and the eukaryotic cytosol. In this study, we have constructed the asymmetric ring complex of a group II chaperonin using circular permutated covalent mutants. Although one ring of the asymmetric ring complex lacks ATPase or ATP binding activity, the other wild-type ring undergoes an ATP-dependent conformational change and maintains protein-folding activity. The results clearly demonstrate that inter-ring communication is dispensable in the reaction cycle of group II chaperonins.     

A catalytic defect in mitochondrial respiratory chain complex I due to a mutation in NDUFS2 in a patient with Leigh syndrome

In this study, we investigated the pathogenicity of a homozygous Asp446Asn mutation in the NDUFS2 gene of a patient with a mitochondrial respiratory chain complex I deficiency. The clinical, biochemical, and genetic features of the NDUFS2 patient were compared with those of 4 patients with previously identified NDUFS2 mutations. All 5 patients presented with Leigh syndrome. In addition, 3 out of 5 showed hypertrophic cardiomyopathy. Complex I amounts in the patient carrying the Asp446Asn mutation were normal, while the complex I activity was strongly reduced, showing that the NDUFS2 mutation affects complex I enzymatic function. By contrast, the 4 other NDUFS2 patients showed both a reduced amount and activity of complex I. The enzymatic defect in fibroblasts of the patient carrying the Asp446Asn mutation was rescued by transduction of wild type NDUFS2. A 3-D model of the catalytic core of complex I showed that the mutated amino acid residue resides near the coenzyme Q binding pocket. However, the K_M of complex I for coenzyme Q analogs of the Asp446Asn mutated complex I was similar to the K_M observed in other complex I defects and in controls. We propose that the mutation interferes with the reduction of coenzyme Q or with the coupling of coenzyme Q reduction with the conformational changes involved in proton pumping of complex I.     

Measurement of Lifespan in Drosophila melanogaster

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced physical performance and increased risk of disease. Individual aging is manifest at the population level as an increase in age-dependent mortality, which is often measured in the laboratory by observing lifespan in large cohorts of age-matched individuals. Experiments that seek to quantify the extent to which genetic or environmental manipulations impact lifespan in simple model organisms have been remarkably successful for understanding the aspects of aging that are conserved across taxa and for inspiring new strategies for extending lifespan and preventing age-associated disease in mammals.The vinegar fly, Drosophila melanogaster, is an attractive model organism for studying the mechanisms of aging due to its relatively short lifespan, convenient husbandry, and facile genetics. However, demographic measures of aging, including age-specific survival and mortality, are extraordinarily susceptible to even minor variations in experimental design and environment, and the maintenance of strict laboratory practices for the duration of aging experiments is required. These considerations, together with the need to practice careful control of genetic background, are essential for generating robust measurements. Indeed, there are many notable controversies surrounding inference from longevity experiments in yeast, worms, flies and mice that have been traced to environmental or genetic artifacts^1-4. In this protocol, we describe a set of procedures that have been optimized over many years of measuring longevity in Drosophila using laboratory vials. We also describe the use of the dLife software, which was developed by our laboratory and is available for download (http://sitemaker.umich.edu/pletcherlab/software). dLife accelerates throughput and promotes good practices by incorporating optimal experimental design, simplifying fly handling and data collection, and standardizing data analysis. We will also discuss the many potential pitfalls in the design, collection, and interpretation of lifespan data, and we provide steps to avoid these dangers.     

Regulation of the activity of the tumor suppressor PTEN by thioredoxin in Drosophila melanogaster

Human Thioredoxin-1 (hTrx-1) is a small redox protein with a molecular weight of 12 kDa that contains two cysteine residues found in its catalytic site. HTrx-1 plays an important role in cell growth, apoptosis, and cancer patient prognosis. Recently, we have demonstrated that hTrx-1 binds to the C2 domain of the human tumor suppressor, PTEN, in a redox dependent manner. This binding leads to the inhibition of PTEN lipid phosphatase activity in mammalian tissue culture systems. In this study, we show that over-expression of hTrx-1 in Drosophila melanogaster promotes cell growth and proliferation during eye development as measured by eye size and ommatidia size. Furthermore, hTrx-1 rescues the small eye phenotype induced by the over-expression of PTEN. We demonstrate that this rescue of the PTEN-induced eye size phenotype requires cysteine-218 in the C2 domain of PTEN. We also show that hTrx-1 over-expression results in increased Akt phosphorylation in fly head extracts supporting our observations that the hTrx-1-induced eye size increase results from the inhibition of PTEN activity. Our study confirms the redox regulation of PTEN through disulfide bond formation with the hTrx-1 in Drosophila and suggests conserved mechanisms for thioredoxins and their interactions with the phosphatidylinositol-3-kinase signaling pathway in humans and fruit flies.     

Ecdysteroid control of metamorphosis in the differentiating adult leg structures of Drosophila melanogaster

During insect metamorphosis, the steroid hormone 20-hydroxyecdysone (20E) is responsible for coordinating the differentiation of adult structures. Several structures of the Drosophila melanogaster adult leg, the six distalmost joints, the bristles, and the pretarsal claws, were examined to investigate how 20E controls their development in vitro. Joints, bristles, and claws were dependent on 20E for differentiation between 20-22 and 24-26 h after puparium formation (APF). After 26-28 h APF, differentiation became hormone independent. Tissue-specific markers in 20E-free cultures showed that the bristle and joint cells had not undergone any further morphogenetic progression. In contrast, the pretarsi underwent partial differentiation. The concentration of 20E required for differentiation was structure specific; tarsal joints required higher concentrations of 20E (greater than 400 ng 20 E/ml) than pretarsal claws, bristles, and other joints (greater than 40 ng 20E/ml). The 20E precursor ecdysone (E) was also able to induce differentiation at concentrations over 700 ng E/ml, but did not show any synergistic interactions with 20E. Lastly, leg structures had a finite ability to respond to 20E; tarsal joints lost competence to respond after 32-34 h APF, while the remaining structures became incompetent after 44-46 h APF.     

Sex-Specific Effects of Cis-Regulatory Variants in Drosophila melanogaster

Sexual dimorphism at the level of gene expression is common and well documented, but much less is known about how different cis-regulatory alleles interact with the different trans-regulatory environments present in males and females. Here we show that sex-specific effects of cis-regulatory variants are common in Drosophila.     

Charge-Rich Regions Modulate the Anti-Aggregation Activity of Hsp90

Protein aggregation can have dramatic effects on cellular function and plays a causative role in many human diseases. In all cells, molecular chaperones bind to aggregation-prone proteins and hinder aggregation. The ability of a protein to resist aggregation and remain soluble in aqueous solution is linked to the physical properties of the protein. Numerous physical studies demonstrate that charged atoms favor solubility. We note that many molecular chaperones possess a substantial negative charge that may allow them to impart solubility on aggregation-prone proteins. Hsp90 is one such negatively charged molecular chaperone. The charge on Hsp90 is largely concentrated in two highly acidic regions. To investigate the relationship between chaperone charge and protein solubility, we deleted these charge-rich regions and analyzed the resulting Hsp90 constructs for anti-aggregation activity. We found that deletion of both charge-rich regions dramatically impaired Hsp90 anti-aggregation activity. The anti-aggregation role of the deleted charge-rich regions could be due to net charge or sequence-specific features. To distinguish these possibilities, we attached an acid-rich region with a distinct amino acid sequence to our double-deleted Hsp90 construct. This charge rescue construct displayed effective anti-aggregation activity indicating that the net charge of Hsp90 contributes to its anti-aggregation activity.     

Automated identification of social interaction criteria in Drosophila melanogaster

The study of social behaviour within groups has relied on fixed definitions of an ‘interaction’. Criteria used in these definitions often involve a subjectively defined cut-off value for proximity, orientation and time (e.g. courtship, aggression and social interaction networks) and the same numerical values for these criteria are applied to all of the treatment groups within an experiment. One universal definition of an interaction could misidentify interactions within groups that differ in life histories, study treatments and/or genetic mutations. Here, we present an automated method for determining the values of interaction criteria using a pre-defined rule set rather than pre-defined values. We use this approach and show changing social behaviours in different manipulations of Drosophila melanogaster. We also show that chemosensory cues are an important modality of social spacing and interaction. This method will allow a more robust analysis of the properties of interacting groups, while helping us understand how specific groups regulate their social interaction space.     

Genetic analysis of mitochondrial protein misfolding in Drosophila melanogaster

Protein misfolding has a key role in several neurological disorders including Parkinson's disease. Although a clear mechanism for such proteinopathic diseases is well established when aggregated proteins accumulate in the cytosol, cell nucleus, endoplasmic reticulum and extracellular space, little is known about the role of protein aggregation in the mitochondria. Here we show that mutations in both human and fly PINK1 result in higher levels of misfolded components of respiratory complexes and increase in markers of the mitochondrial unfolded protein response. Through the development of a genetic model of mitochondrial protein misfolding employing Drosophila melanogaster, we show that the in vivo accumulation of an unfolded protein in mitochondria results in the activation of AMP-activated protein kinase-dependent autophagy and phenocopies of pink1 and parkin mutants. Parkin expression acts to clear mitochondria with enhanced levels of misfolded proteins by promoting their autophagic degradation in vivo, and refractory to Sigma P (ref(2)P), the Drosophila orthologue of mammalian p62, is a critical downstream effector of this quality control pathway. We show that in flies, a pathway involving pink1, parkin and ref(2)P has a role in the maintenance of a viable pool of cellular mitochondria by promoting organellar quality control.     

Ether-induced segmentation disturbances in Drosophila melanogaster

Summary Drosophila embryos, exposed to ether between 1 and 4 h after oviposition, develop defects ranging from the complete lack of segmentation to isolated gaps in single segments. Between these extremes are varying extents of incomplete and abnormal segmentation. On the basis of both their temporal and spatial characteristics, five major phenotype classes may be distinguished: headless — unsegmented or incompletely segmented anteriorly; gap — interruptions of segmentation not obviously periodic; alternating segment gaps — interruptions with double segment periodicities; fused segments; and short segments — truncations with single segment periodicities. Many defects resemble known mutant phenotypes. The disturbances in segmentation are predominantly global and frequently accompanied by alterations in segment specification, such that the segments obtained show no resemblance to the normal homologues. These features, together with the distinctive spatiotemporal characteristics of the defects, all point to segmentation as a dynamic process. The regular spacing of the segments and the fact that the entire range of defects is inducible by ether are further consistent with the hypothesis that at least part of the segmentation process may consist of physicochemical reactions coordinated over the whole body. The relationship between our data and data from genetic and other analyses are briefly discussed.     

Metabolic effects of CO2 anaesthesia in Drosophila melanogaster

Immobilization of insects is necessary for various experimental purposes, and CO₂ exposure remains the most popular anaesthetic method in entomological research. A number of negative side effects of CO₂ anaesthesia have been reported, but CO₂ probably brings about metabolic modifications that are poorly known. In this work, we used GC/MS-based metabolic fingerprinting to assess the effect of CO₂ anaesthesia in Drosophila melanogaster adults. We analysed metabolic variation of flies submitted to acute CO₂ exposure and assessed the temporal metabolic changes during short- and long-term recovery. We found that D. melanogaster metabotypes were significantly affected by the anaesthetic treatment. Metabolic changes caused by acute CO₂ exposure were still manifested after 14 h of recovery. However, we found no evidence of metabolic alterations when a long recovery period was allowed (more than 24 h). This study points to some metabolic pathways altered during CO₂ anaesthesia (e.g. energetic metabolism). Evidence of short-term metabolic changes indicates that CO₂ anaesthesia should be used with utmost caution in physiological studies when a short recovery is allowed. In spite of this, CO₂ treatment seems to be an acceptable anaesthetic method provided that a long recovery period is allowed (more than 24 h).     

Evolution of foraging behaviour in response to chronic malnutrition in Drosophila melanogaster

Chronic exposure to food of low quality may exert conflicting selection pressures on foraging behaviour. On the one hand, more active search behaviour may allow the animal to find patches with slightly better, or more, food; on the other hand, such active foraging is energetically costly, and thus may be opposed by selection for energetic efficiency. Here, we test these alternative hypotheses in Drosophila larvae. We show that populations which experimentally evolved improved tolerance to larval chronic malnutrition have shorter foraging path length than unselected control populations. A behavioural polymorphism in foraging path length (the rover-sitter polymorphism) exists in nature and is attributed to the foraging locus (for). We show that a sitter strain (for(s2)) survives better on the poor food than the rover strain (for(R)), confirming that the sitter foraging strategy is advantageous under malnutrition. Larvae of the selected and control populations did not differ in global for expression. However, a quantitative complementation test suggests that the for locus may have contributed to the adaptation to poor food in one of the selected populations, either through a change in for allele frequencies, or by interacting epistatically with alleles at other loci. Irrespective of its genetic basis, our results provide two independent lines of evidence that sitter-like foraging behaviour is favoured under chronic larval malnutrition.     

Glial cells phagocytose neuronal debris during the metamorphosis of the central nervous system in Drosophila melanogaster

 Using electron microscopy we demonstrate that degenerating neurons and cellular debris resulting from neuronal reorganization are phagocytosed by glial cells in the brain and nerve cord of the fruitfly Drosophila melanogaster during the first few hours following pupariation. At this stage several classes of glial cells appear to be engaged in intense phagocytosis. In the cell body rind, neuronal cell bodies are engulfed and phagocytosed by the same glial cells that enwrap healthy neurons in this region. In the neuropil, cellular debris in tracts and synaptic centres resulting from metamorphic re-differentiation of larval neurons is phagocytosed by neuropil-associated glial cells. Phagocytic glial cells are hypertrophied, produce large amounts of lysosome-like bodies and contain a large number of mitochondria, condensed chromatin bodies, membranes and other remains from neuronal degeneration in phagosomes. Received: 23 January 1996 / Accepted in revised form: 21 May 1996     

Genetic basis of natural variation in body pigmentation in Drosophila melanogaster

Body pigmentation in insects and other organisms is typically variable within and between species and is often associated with fitness. Regulatory variants with large effects at bab1, t and e affect variation in abdominal pigmentation in several populations of Drosophila melanogaster. Recently, we performed a genome wide association (GWA) analysis of variation in abdominal pigmentation using the inbred, sequenced lines of the Drosophila Genetic Reference Panel (DGRP). We confirmed the large effects of regulatory variants in bab1, t and e; identified 81 additional candidate genes; and validated 17 candidate genes (out of 28 tested) using RNAi knockdown of gene expression and mutant alleles. However, these analyses are imperfect proxies for the effects of segregating variants. Here, we describe the results of an extreme quantitative trait locus (xQTL) GWA analysis of female body pigmentation in an outbred population derived from light and dark DGRP lines. We replicated the effects on pigmentation of 28 genes implicated by the DGRP GWA study, including bab1, t and e and 7 genes previously validated by RNAi and/or mutant analyses. We also identified many additional loci. The genetic architecture of Drosophila pigmentation is complex, with a few major genes and many other loci with smaller effects.     

High-resolution Quantification of Odor-guided Behavior in Drosophila melanogaster Using the Flywalk Paradigm

In their natural environment, insects such as the vinegar fly Drosophila melanogaster are bombarded with a huge amount of chemically distinct odorants. To complicate matters even further, the odors detected by the insect nervous system usually are not single compounds but mixtures whose composition and concentration ratios vary. This leads to an almost infinite amount of different olfactory stimuli which have to be evaluated by the nervous system.To understand which aspects of an odor stimulus determine its evaluation by the fly, it is therefore desirable to efficiently examine odor-guided behavior towards many odorants and odor mixtures. To directly correlate behavior to neuronal activity, behavior should be quantified in a comparable time frame and under identical stimulus conditions as in neurophysiological experiments. However, many currently used olfactory bioassays in Drosophila neuroethology are rather specialized either towards efficiency or towards resolution.Flywalk, an automated odor delivery and tracking system, bridges the gap between efficiency and resolution. It allows the determination of exactly when an odor packet stimulated a freely walking fly, and to determine the animal´s dynamic behavioral reaction.