No abstract

The roles that the pineal gland and its hormone melatonin play in the regulation of circadian rhythmicity and photoperiodism vary among vertebrate species. Recently, putative sites of melatonin action have been elucidated in several avian and mammalian species by application of in vitro binding of a radioiodinated melatonin agonist, 2[¹²⁵I]iodomeIatonin (IMEL) and autoradioradiography. These studies in mammals, birds and reptiles have indicated profound differences in the distribution of IMEL binding between these diverse groups, suggesting that these large differences in binding may reflect differences in melatonin function. The present study was performed to determine systematically whether the variance in IMEL binding among avian species corresponds to changes in circadian organization and/or phylogenetic relationships. The distribution of specific IMEL binding was determined in the brains from birds belonging to 14 different species in 5 Orders (Psittaciformes, Passeriformes, Columbiformes, Galliformes and Anseriformes) using in vitro binding, autoradiography and computer-assisted image analysis. The distribution was compared to a similar study in 3 species of turtles as an outgroup. The data indicated IMEL binding in retinorecipient structures of the circadian, tectofugal, thalamofugal and accessory optic visual pathways in all avian species. Relay nuclei and integrative structures of the tectofugal, thalamofugal, accessory optic, and limbic systems, however, bound the hormone to varying degrees. In turtles, binding was observed in retinorecipient structures of the thalamofugal visual pathway and in retinorecipient and integrative areas of the tectofugal visual pathway. No binding was observed in the pineal gland, tuberal hypothalamus or adenohypophysis in any avian or testudine species. This distribution is drastically different from that observed in mammals, where binding predominates in the pars tuberalis of the adenohypophysis and in the suprachiasmatic nucleus, suggesting that the circadian system may influence a wide array of sensory and integrative functions in birds and reptiles through the circadian secretion of melatonin, but that this capacity has been lost in mammals.

An efficient and low-cost method of examining larval movement in Drosophila melanogaster is needed to study how mutations and/or alterations in the muscular, neural, and olfactory systems affect locomotor behavior. Here, we describe the implementation of wrMTrck, a freely available ImageJ plugin originally developed for examining multiple behavioral parameters in the nematode C. elegans. Our optimized method is rapid, reproducible and does not require automated microscope setups or the purchase of proprietary software. To demonstrate the utility of this method, we analyzed the velocity and crawling paths of two Drosophila mutants that affect muscle structure and/or function. Additionally, we show that this approach is useful for tracking the behavior of adult insects, including Tribolium castaneum and Drosophila melanogaster.

The pineal gland and its hormone melatonin are important for the generation of circadian rhythms by passerine birds such as the house sparrow, Passer domesticus. The sites of melatonin action within the brain of this species were determined by employing two techniques. First, the distribution of 2[125iliodomelatonin (IMEL) binding was determined by in vitro incubation in IMEL, autoradiography, and computer image analysis. Data from these experiments indicated reversible IMEL binding in a wide array of cerebral structures primarily associated with vision. Second, the effects of exogenous melatonin on cerebral uptake of the metabolic marker 2-deoxy['4Clglucose (2DG) were determined. Many of the same structures that bind IMEL also exhibited decreased 2DG uptake in response to melatonin administration, whereas structures that did not bind IMEL were unaffected by the hormone. The data suggest that much of the house sparrow's visual world is modulated on a circadian basis via the circadian secretion of melatonin. These observations are discussed in the context of avian circadian organization and the role it may play in the behavioral physiology of the bird.

Cell growth and/or proliferation may require the reprogramming of metabolic pathways, whereby a switch from oxidative to glycolytic metabolism diverts glycolytic intermediates towards anabolic pathways. Herein, we identify a novel role for TRIM32 in the maintenance of glycolytic flux mediated by biochemical interactions with the glycolytic enzymes Aldolase and Phosphoglycerate mutase. Loss of Drosophila TRIM32, encoded by thin (tn), shows reduced levels of glycolytic intermediates and amino acids. This altered metabolic profile correlates with a reduction in the size of glycolytic larval muscle and brain tissue. Consistent with a role for metabolic intermediates in glycolysis-driven biomass production, dietary amino acid supplementation in tn mutants improves muscle mass. Remarkably, TRIM32 is also required for ectopic growth - loss of TRIM32 in a wing disc-associated tumor model reduces glycolytic metabolism and restricts growth. Overall, our results reveal a novel role for TRIM32 for controlling glycolysis in the context of both normal development and tumor growth.

The inability to remove protein aggregates in post-mitotic cells such as muscles or neurons is a cellular hallmark of aging cells and is a key factor in the initiation and progression of protein misfolding diseases. While protein aggregate disorders share common features, the molecular level events that culminate in abnormal protein accumulation cannot be explained by a single mechanism. Here we show that loss of the serine/threonine kinase NUAK causes cellular degeneration resulting from the incomplete clearance of protein aggregates in Drosophila larval muscles. In NUAK mutant muscles, regions that lack the myofibrillar proteins F-actin and Myosin heavy chain (MHC) instead contain damaged organelles and the accumulation of select proteins, including Filamin (Fil) and CryAB. NUAK biochemically and genetically interacts with Drosophila Starvin (Stv), the ortholog of mammalian Bcl-2-associated athanogene 3 (BAG3). Consistent with a known role for the co-chaperone BAG3 and the Heat shock cognate 71 kDa (HSC70)/HSPA8 ATPase in the autophagic clearance of proteins, RNA interference (RNAi) of Drosophila Stv, Hsc70-4, or autophagy-related 8a (Atg8a) all exhibit muscle degeneration and muscle contraction defects that phenocopy NUAK mutants. We further demonstrate that Fil is a target of NUAK kinase activity and abnormally accumulates upon loss of the BAG3-Hsc70-4 complex. In addition, Ubiquitin (Ub), ref(2)p/p62, and Atg8a are increased in regions of protein aggregation, consistent with a block in autophagy upon loss of NUAK. Collectively, our results establish a novel role for NUAK with the Stv-Hsc70-4 complex in the autophagic clearance of proteins that may eventually lead to treatment options for protein aggregate diseases.

Control of tissue and organismal size requires the continual reprogramming of metabolic pathways to integrate biosynthetic and degradative signals. During cell growth and/or proliferation, one such mechanism that promotes the accumulation of cellular material is a switch from oxidative to glycolytic metabolism, whereby glycolytic intermediates are diverted towards anabolic pathways. How this switch is regulated in different tissues is not clear. Herein we identify a novel role for the tripartite motif (TRIM) family member, TRIM32, in the maintenance of glycolytic flux. Using a proteomics approach, we uncovered the glycolytic enzymes Aldolase (Ald) and Phosphoglycerate mutase 78 (Pglym) as TRIM32 interacting proteins. Loss of Drosophila TRIM32, encoded by the thin (tn) gene, showed a reduction in glycolytic activity and amino acid abundance. This altered metabolic profile caused a striking reduction in the overall size of two inherently glycolytic larval tissues – somatic muscles and the developing brain. Consistent with a role for metabolic intermediates in glycolysis‐driven biomass production, nutritional supplementation of amino acids in tn mutants restored muscle mass. Many tumors favor glycolytic metabolism to maximize substrate production for uncontrolled cell growth and proliferation. Remarkably, wing disc‐associated tumor growth is abolished upon loss of TRIM32. Our results reveal a novel connection between TRIM32 and the maintenance of glycolytic enzyme levels and upregulated pathway activity for the sustained growth of normal and cancerous tissue growth. Support or Funding Information This work was supported by a grant through the National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) to E.R.G (R01AR060788). J.M.T. is supported by a MIRA award from NIGMS (R35GM119557).

Expression of human disease-causing LGMD2H alleles in Drosophila causes muscle degeneration and elevated levels of integrin and sarcoglycan costamere proteins, likely exacerbating disease progression.