Copper homoeostasis in Drosophila melanogaster S2 cells

Abstract

Copper homoeostasis was investigated in the Drosophila melanogaster S2 cell line to develop an insect model for the study of copper regulation. Real-time PCR studies have demonstrated expression in S2 cells of putative orthologues of human Cu regulatory genes involved in the uptake, transport, sequestration and efflux of Cu. Drosophila orthologues of the mammalian Cu chaperones, ATOX1 (a human orthologue of yeast ATX1), CCS (copper chaperone for superoxide dismutase), COX17 (a human orthologue of yeast COX17), and SCO1 and SCO2, did not significantly respond transcriptionally to increased Cu levels, whereas MtnA, MtnB and MtnD (Drosophila orthologues of human metallothioneins) were up-regulated by Cu in a time- and dose-dependent manner. To examine the effect on Cu homoeostasis, expression of several key copper homoeostasis genes was suppressed using double-stranded RNA interference. Suppression of the MTF-1 (metal-regulatory transcription factor 1), reduced both basal and Cu-induced gene expressions of MtnA, MtnB and MtnD, significantly reducing the tolerance of these cells to increased Cu. Suppression of either Ctr1A (a Drosophila orthologue of yeast CTR1) or Ctr1B significantly reduced Cu uptake from media, demonstrating that both these proteins function to transport Cu into S2 cells. Significantly, Cu induced Ctr1B gene expression, and this could be prevented by suppressing MTF-1, suggesting that Ctr1B might be involved in Cu detoxification. Suppression of DmATP7, the putative homologue of human Cu transporter genes ATP7A and ATP7B, significantly increased Cu accumulation, demonstrating that DmATP7 is essential for efflux of excess Cu. This work is consistent with previous studies in mammalian cells, validating S2 cells as a model system for studying Cu transport and identifying novel Cu regulatory mechanisms.

Malvolio is a copper transporter in Drosophila melanogaster

“…Mechanisms for uptake, distribution, sequestration and efflux of Cu are well conserved among yeast, insect and mammalian systems (Camakaris et al, 1999). The Ctr family are the best characterised Cu uptake proteins, with Drosophila melanogaster having three, designated Ctr1A, B and C. In vivo and in vitro studies have demonstrated Ctr1A and Ctr1B are functional Cu transporters with different tissue and life stage expression profiles (Southon et al, 2004;Turski and Thiele, 2007;Zhou et al, 2003). Although human CTR1 (also known as SLC31A1 -HUGO) has traditionally been seen as the primary Cu uptake mechanism in gut epithelium, a recent study by Zimnicka et al (Zimnicka et al, 2007) has found endogenous CTR1 to be localised to the basolateral surface of cultured intestinal and kidney cells, rather than the apical membrane, suggesting CTR1 may not be responsible for dietary Cu absorption.…”

Formulation of a protein-free medium based on IPL-41 for the sustained growth of Drosophila melanogaster S2 cells

“…Drosophila melanogaster cells are often maintained in Schneider's Drosophila medium supplemented with 10% fetal bovine serum, but this cell line can be also cultivated in formulations such as Shields and Sang M3 Insect Medium containing from 2% to 10% FBS (Mosher and Crews 1999;Nybakken et al 2005) and in Drosophila Serum-Free medium (Southon et al 2004). The last medium composition, however, similarly to that of Sf900II medium (a culture medium frequently employed for insect cell culture), is not directly available in the literature.…”

The Intestinal Copper Exporter CUA-1 Is Required for Systemic Copper Homeostasis in Caenorhabditis elegans

“…For these reasons, we utilized C. elegans, which has been widely used in the study of the metabolism of other metals, including iron, heme, and zinc (8,(13)(14)(15)(16)(17). Although mammals have two copper exporters, serving complementary functions in different tissues such as the gut and liver, lower metazoans (including nematodes (C. elegans) and insects (Drosophila melanogaster)) have only a single homolog of ATP7A/B (2,18,19). The protein product of C. elegans cua-1 shares very high sequence similarity with human ATP7A/B.…”