Real-time study of spatio-temporal dynamics (4D) of physiological activities in alive biological specimens with different FOVs and resolutions simultaneously.

This article reports the development of a microscopy imaging system that gives feasibility for studying spatio-temporal dynamics of physiological activities of alive biological specimens (over entire volume not only for a particular section, i.e., in 4D). The imaging technology facilitates to obtain two image frames of a section of the larger specimen ([Formula: see text]) with different FOVs at different resolutions or magnifications simultaneously in real-time (in addition to recovery of 3D (volume) information). Again, this imaging system addresses the longstanding challenges of housing multiple light sources (6 at the maximum till date) in microscopy (in general) and light sheet fluorescence microscopy (LSFM) (in particular), by using a tuneable pulsed laser source (with an operating wavelength in the range [Formula: see text]-670 nm) in contrast to the conventional CW laser source being adopted for inducing photo-excitation of tagged fluorophores. In the present study, we employ four wavelengths ([Formula: see text] 488 nm, 585 nm, 590 nm, and 594 nm). Our study also demonstrates quantitative characterization of spatio-temporal dynamics (velocity-both amplitude and direction) of organelles (mitochondria) and their mutual correlationships. Mitochondria close to the nucleus (or in clustered cells) are observed to possess a lower degree of freedom in comparison to that at the cellular periphery (or isolated cells). In addition, the study demonstrates real-time observation and recording of the development and growth of all tracheal branches during the entire period ([Formula: see text] min) of embryonic development (Drosophila). The experimental results-with experiments being conducted in various and diversified biological specimens (Drosophila melanogaster, mouse embryo, and HeLa cells)-demonstrate that the study is of great scientific impact both from the aspects of technology and biological sciences.

Sexually dimorphic microRNA miR-190 regulates lifespan in male Drosophila.

microRNAs are short noncoding RNAs that buffer fluctuations in gene expression in a myriad of physiological conditions. Here, we carried out a screen to identify the role of microRNAs in the maintenance of age-dependent neuronal functions in adult Drosophila. We report that miR-190 acts in the neurons to regulate lifespan, neuronal maintanence and age-related locomotor activity specifically in male flies. miR-190, a highly conserved microRNA, shows higher expression levels in male flies. Our data suggest that miR-190 functions by regulating target genes that are involved in maintaining neuronal activity and lifespan in male flies.

Nutrient dependent miR-184 aids in the survival of Drosophila larvae during low food conditions.

Nutrition is one of the critical factors known to regulate the development and growth of organisms. Lack of nutrients affects the proper functioning and survival of organisms. However, fluctuation of the levels of nutrients is quite common in a natural environment, and organisms have evolved various molecular and physiological means by which they can survive such conditions. microRNAs are short non-coding RNAs that play significant biological functions, primarily by acting as post-transcriptional buffers of noisy gene expression. Recent studies show that miR-184, a conserved microRNA, is expressed at higher levels in low nutrition conditions. Our experiments show that miR-184 mutants showed enhanced lethality when raised in low nutrient food conditions. Here, we demonstrate the role of miR-184, a microRNA regulated by nutritional status, also helps in the survival of the larvae to adulthood in low food conditions.

Edem1 activity in the fat body regulates insulin signalling and metabolic homeostasis in Drosophila.

In Drosophila, nutrient status is sensed by the fat body, a functional homolog of mammalian liver and white adipocytes. The fat body conveys nutrient information to insulin-producing cells through humoral factors which regulate Drosophila insulin-like peptide levels and insulin signalling. Insulin signalling has pleiotropic functions, which include the management of growth and metabolic pathways. Here, we report that Edem1 (endoplasmic reticulum degradation-enhancing α-mannosidase-like protein 1), an endoplasmic reticulum-resident protein involved in protein quality control, acts in the fat body to regulate insulin signalling and thereby the metabolic status in Drosophila Edem1 limits the fat body-derived Drosophila tumor necrosis factor-α Eiger activity on insulin-producing cells and maintains systemic insulin signalling in fed conditions. During food deprivation, edem1 gene expression levels drop, which aids in the reduction of systemic insulin signalling crucial for survival. Overall, we demonstrate that Edem1 plays a vital role in helping the organism to endure a fluctuating nutrient environment by managing insulin signalling and metabolic homeostasis.

Lint, a transmembrane serine protease, regulates growth and metabolism in Drosophila.

Insulin signaling in Drosophila has a significant role in regulating growth, metabolism, fecundity, stress response, and longevity. The molecular mechanism by which insulin signaling regulates these vital processes is dependent on the nutrient status and oxygen availability of the organism. In a genetic screen to identify novel genes that regulate Drosophila insulin signaling, we discovered lumens interrupted (lint), a gene that has previously been shown to act in tracheal development. The knockdown of lint gene expression using a Dilp2Gal4 driver which expresses in the neuronal insulin producing cells (IPCs), led to defects in systemic insulin signaling, metabolic status and growth. However, our analysis of lint knockdown phenotypes revealed that downregulation of lint in the trachea and not IPCs was responsible for the growth phenotypes, as the Gal4 driver is also expressed in the tracheal system. We found various tracheal terminal branch defects, including reduction in the length as well as number of branches in the lint knockdown background. Our study reveals that substantial effects of lint downregulation arose because of tracheal defects, which induced tissue hypoxia, altered systemic insulin/TOR signaling, and resulted in effects on developmental growth regulation.

Larval nutrition influences adult fat stores and starvation resistance in Drosophila.

Insulin plays a major role in connecting nutrient availability to energy homeostasis by regulating metabolic pathways. Defects in insulin signalling is the primary cause for diabetes, obesity and various metabolic disorders. Nutritional status during growth and developmental stages play a crucial role in determining adult size, fecundity and ageing. However, the association between developmental nutrition and adult metabolic disorders has not been fully explored. Here, we address the effects of nutrient status during the larval growth phase on adult metabolism in Drosophila. We report that restricted food supply in larvae led to higher fat reserves and starvation resistance in mature adult flies, which we attribute to low insulin signalling. A lesser amount of stored fat was mobilised during early adult stages and during acute starvation, which accounts for the metabolic effects. Furthermore, larval diet influenced the expression of fat mobilisation genes brummer and lipid storage droplet-2 in adult flies, which led to the metabolic phenotypes reported here. Thus, the restricted nutrient environment in developing larvae led to adaptive changes that entrain the adult flies for scarce food availability.

Insulin signalling elicits hunger-induced feeding in Drosophila.

Insulin, a highly conserved peptide hormone, links nutrient availability to metabolism and growth in animals. In fed states insulin levels remain high and in animals that are food deprived insulin signalling drops. Here, we report that in Drosophila, feeding elicited by short periods of starvation is dependent on insulin signalling. The activity of insulin signalling pathway in the abdominal fatbody aids in feeding during short periods of starvation. A feedback regulatory signalling that involves cells that express the Drosophila hunger hormone short-neuropeptide-F (sNPF) and insulin-producing cells sustain the orexigenic function of insulin. Furthermore, the orexigenic phase of insulin activity aids in the efficient management of nutrient stores and survival of flies during starvation.

Achiasmy: Male Fruit Flies Are Not Ready to Mix.

Maintenance of the chromosomal copy number over generations and recombination between homologous chromosomes are hallmarks of meiotic cell division. This genetic exchange that take place during gamete formation leads to genetic diversity, the main driving force behind natural selection. Formation of chiasmata, the physical link between homologous chromosomes during meiosis, is a requisite for recombination. In addition, chiasmata also aid in proper segregation of homologous chromosomes and has a major impact on reproductive fitness. Given these facts it is intriguing that many insect species have forgone the need for genetic exchange between homologous chromosomes during meiosis. Geneticists for several decades knew that meiotic crossover and recombination is absent in Drosophila males and some female lepidopterans, a condition termed achiasmy. However, a good understanding of the mechanisms that cause achiasmy and the evolutionary benefits of achiasmy is currently lacking. In this article we will discuss possible genetic and molecular basis of achiasmy in male Drosophila.

Drosophila miR-14 regulates insulin production and metabolism through its target, sugarbabe.

Energy homeostasis depends on insulin signaling in metazoans. Insulin levels reflect the nutritional status of the animal to control levels of circulating sugar and regulate storage of resources in the form of glycogen and fat. Over the past several years, evidence has begun to accumulate that insulin production and secretion, as well as cellular responsiveness to insulin, are subject to regulation by microRNAs. Here we present evidence that miR-14 acts in the insulin-producing neurosecretory cells in the adult Drosophila brain to control metabolism. miR-14 acts in these cells through its direct target, sugarbabe. sugarbabe encodes a predicted zinc finger protein that regulates insulin gene expression in the neurosecretory cells. Regulation of sugarbabe levels by nutrients and by miR-14 combines to allow the fly to manage resource mobilization in a nutritionally variable environment.

microRNA miR-14 acts to modulate a positive autoregulatory loop controlling steroid hormone signaling in Drosophila.

The insect steroid hormone Ecdysone and its receptor play important roles during development and metamorphosis and regulate adult physiology and life span. Ecdysone signaling, via the Ecdysone receptor (EcR), has been proposed to act in a positive autoregulatory loop to increase EcR levels and sensitize the animal to ecdysone pulses. Here we present evidence that this involves EcR-dependent transcription of the EcR gene, and that the microRNA miR-14 modulates this loop by limiting expression of its target EcR. Ecdysone signaling, via EcR, down-regulates miR-14. This alleviates miR-14-mediated repression of EcR and amplifies the response. Failure to limit EcR levels is responsible for the many of the defects observed in miR-14 mutants. miR-14 plays a key role in modulating the positive autoregulatory loop by which Ecdysone sensitizes its own signaling pathway.

Head involution defective (Hid)-triggered apoptosis requires caspase-8 but not FADD (Fas-associated death domain) and is regulated by Erk in mammalian cells.

The molecular machinery of apoptosis is evolutionarily conserved with some exceptions. One such example is the Drosophila proapoptotic gene Head involution defective (Hid), whose mammalian homologue is not known. Hid is apoptotic to mammalian cells, and we have examined the mechanism by which Hid induces death. We demonstrate for the first time a role for the extracellular signal-related kinase-1/2 (Erk-1/2) in the regulation of Hid function in mammalian cells. Bcl-2 and an inhibitor of caspase-9 blocked apoptosis, indicative of a role for the mitochondrion in this pathway, and we provide evidence for a role for caspase-8 in Hid-induced apoptosis. Thus, apoptosis was blocked by an inhibitor of caspase-8, deletion of caspase-8 rendered cells resistant to Hid-induced apoptosis, and Hid associated with caspase-8 in cell lysates. The Fas-associated death domain (FADD) was dispensable for the apoptotic function of Hid, indicating that Hid does not require extracellular death receptor signaling for the activation of caspase-8. In activated T cells, the cytokine interleukin-2 blocked caspase-8 processing and apoptosis, suggesting that survival cues from trophic factors may target a Hid-like intermediate present in mammalian cells. Thus, this study shows that Hid engages with conserved components of cellular death machinery and suggests that apoptotic paradigms characterized by FADD-independent activation of caspase-8 may involve a Hid-like molecule in mammalian cells.