Abstract
Tissue-specific manipulation of known copper transport genes in Drosophila tissues results in phenotypes that are presumably due to an alteration in copper levels in the targeted cells. However direct confirmation of this has to date been technically challenging. Measures of cellular copper content such as expression levels of copper-responsive genes or cuproenzyme activity levels, while useful, are indirect. First-generation copper-sensitive fluorophores show promise but currently lack the sensitivity required to detect subtle changes in copper levels. Moreover such techniques do not provide information regarding other relevant biometals such as zinc or iron. Traditional techniques for measuring elemental composition such as inductively coupled plasma mass spectroscopy are not sensitive enough for use with the small tissue amounts available in Drosophila research. Here we present synchrotron x-ray fluorescence microscopy analysis of two different Drosophila tissues, the larval wing imaginal disc, and sectioned adult fly heads and show that this technique can be used to detect changes in tissue copper levels caused by targeted manipulation of known copper homeostasis genes.
Highlights
Copper is an essential nutrient required in trace amounts by most organisms
x-ray fluorescence (XRF) elemental maps were collected for wing imaginal discs dissected from wandering third instar Drosophila larvae
The results presented here demonstrate that XRF can be used to rapidly and reliably assess elemental levels in two different Drosophila tissues, the larval imaginal discs and adult eye/head sections
Summary
Copper is an essential nutrient required in trace amounts by most organisms. The main cellular use of copper is as a co-factor for vital copper-dependent enzymes (cuproenzymes) such as Cu, Zn superoxide dismutase, lysyl oxidase, tyrosinase and peptidylglycine a-amidating monooxygenase [1,2,3]. Cellular copper uptake requires the activity of members of the Ctr family of transmembrane proteins which are located at the outer plasma membrane [4,5]. The copper-translocating P-Type ATPase proteins ATP7A (Menkes, MNK) and ATP7B (Wilson Disease, WND) are required for copper transport across the trans Golgi Network membrane into the Golgi lumen for incorporation into cuproenzymes such as tyrosinase [6,7]. ATP7A/B can respond to elevated copper levels by translocating to the outer plasma membrane (PM) to export copper from the cell [9,10]. Copper homeostasis is a balance of copper uptake (Ctr1) and efflux (ATP7A/B) as well as sequestration within the cytoplasm by metallothionein proteins
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