Biology subjects

Filine, E.

Publications and source records attributed to Filine, E..

3 recordsLinked to original sources

bioRxiv2025

Phage display-mediated immuno-PCR to detect low-abundance secreted proteins in Drosophila

Circulating hormones, that mediate communications across organs to maintain physiological balance, are commonly detected and quantified using enzyme-linked immunosorbent assays (ELISAs). However, while ELISA is well-suited for organisms where sample blood can be readily obtained, its application is considerably more challenging in smaller organisms, particularly Drosophila, which has gained widespread use in recent years for physiological studies. Here, we present sensitive phage display-mediated immuno-PCR (PD-iPCR) to detect Drosophila hemolymph proteins via two approaches: 1) by identifying high-affinity nanobodies through phage-display library screening and subsequent affinity maturation and 2) by generating a knock-in fly line producing secreted proteins tagged with tandem NanoTags composed of VHH05 and 127D01. Using these approaches, we successfully established PD-iPCR to detect insulin-binding ImpL2 protein in fly hemolymph. Notably, the tandem NanoTag-based sandwich PD-iPCR enabled highly sensitive detection of tagged antigens, allowing us to quantify elevated ImpL2 levels in the hemolymph of starved flies and those bearing Yki-induced gut tumors. Collectively, our results demonstrate that PD-iPCR enables detection of endogenous, low-abundance circulating hormones in Drosophila, providing a powerful tool for studying interorgan communication. Significance statementHormones and other secreted factors orchestrate organism-wide physiology, yet their routine quantification in Drosophila has been limited by the limited volume of hemolymph available for assays like enzyme-linked immunosorbent assay (ELISA). Here, we present phage display-mediated immuno-PCR (PD-iPCR) as a novel sensitive platform for quantifying secreted proteins in flies in vivo. Using ImpL2 as an example, we successfully detected nanomolar level of circulating ImpL2 and monitored its physiological changes during starvation and tumorigenesis using PD-iPCR. This approach can be readily expanded to multiplexed quantification of secreted proteins in vivo by leveraging the multiple available nanobodies and the vast collection of epitope-tagged Drosophila lines. This work opens the door to systematic endocrine phenotyping across developmental stages and diverse physiological conditions.

genetics↗

bioRxiv2025

Colorectal tumor-induced orexigenic imbalance lowers protein appetite and drives early organ wasting symptoms

Cancer cachexia (CC) is characterized by organ wasting and ensuing involuntary weight loss. Despite advances, underlying mechanisms initiating CC remain unclear, including early symptoms like anorexia. Here, we use a fly gut-tumor model with a precise time-window before organ wasting starts. We show that tumor-induced factors involved in inflammation (unpaired 3/ Interleukin-6-like) and reduced insulin signaling (ImpL2/ Insulin Growth Factor Binding Protein) decrease NPF (Neuropeptide F/ Neuropeptide Y) in the brain prior to organ wasting. This early NPF decrease triggers low protein-specific food appetite and anorexia. We find that ImpL2 reduces NPF signaling while upd3 helps by concurrently disrupting the blood brain barrier. Tumor-induced NPF decrease, and early reduction of protein appetite drive the onset of weight loss and exacerbate the risk of death during organ wasting. Altogether, we provide evidence for an early orexigenic brain imbalance causing low protein appetite that regulates the onset and outcome of organ wasting.

genetics↗

bioRxiv2021

REPTOR/CREBRF encode key regulators of muscle energy metabolism

Metabolic flexibility of muscle tissue describes the capacity to use glucose or lipids as energy substrates and its disruption is associated with metabolic dysfunction. Cancer-induced cachexia is a metabolic syndrome linked with muscle wasting, changes in muscle energy metabolism and lower life expectancy in cancer patients. The molecular mechanisms driving metabolic changes in muscle, however, are poorly characterized. Here, using a Drosophila model of systemic metabolic dysfunction triggered by yorkie-induced gut tumors, we identify the transcription factor REPTOR as a key regulator of energy metabolism in muscle. We show that REPTOR is upregulated in muscles of adult flies with gut yorkie-tumors, where it is necessary to modulate glucose metabolism. REPTOR expression in muscles is induced by ImpL2, a tumor-derived insulin binding protein that reduces systemic insulin signaling, or by nutritional restriction. Further, in vitro and in vivo studies indicate that high activity of REPTOR is sufficient to increase glucose content, transcriptionally repress phosphofructokinase and increase mitochondrial respiration. Consistent with the fly studies, higher levels of CREBRF, the mammalian ortholog of REPTOR, reduce glycolysis in mouse myotubes while promoting an oxidative phenotype. Altogether, our results implicate REPTOR/CREBRF as key regulators of muscle metabolism and metabolic flexibility that share a conserved function as repressors of glycolysis and promoters of oxidative phosphorylation.

physiology↗