Genetics - Theses
Permanent URI for this collection
Search Results
1 - 1 of 1
-
ItemIdentification and characterisation of copper homeostasis genes( 2010)Copper is essential for life, yet also potentially toxic in excess. Copper homeostasis is therefore regulated at the cellular, tissue and organismal levels. Studies with eukaryotic model systems, primarily yeast and mammals, have identified conserved mechanisms for copper uptake, distribution, sequestration and efflux. Nevertheless, there is much we do not know about how copper levels are sensed and regulated. Additional proteins involved in copper homeostasis under both ‘normal’ and ‘diseased’ conditions remain to be identified. The emergence of Drosophila melanogaster as a bona fide model system for the study of copper homeostasis has coincided with research aiming to identify and characterise novel copper regulatory genes in this organism. Whereas others have focused on in vivo studies, the studies reported in this thesis are focussed on cultured D. melanogaster S2 cells. The first aim of this project was to use cDNA microarrays to identify genes transcriptionally regulated by copper levels in S2 cells. The second and third aims were to characterise the function and localisation of novel copper homeostasis genes in vitro and in vivo respectively. Initial characterisation of D. melanogaster S2 cells found these cells express orthologues of key mammalian copper regulatory genes. Copper uptake primarily occurs via Ctr1A and Ctr1B, the orthologues of human Ctr1. Copper efflux occurs via DmATP7, the sole D. melanogaster orthologue of the mammalian P-type ATPases, ATP7A and ATP7B. S2 cells are highly copper tolerant and primarily rely on metallothionein-mediated copper sequestration and copper efflux to maintain homeostasis. Whereas ATP7A and ATP7B undergo copper-induced trafficking between the trans-Golgi network and plasma membrane of mammalian cells, this does not appear to occur with endogenous DmATP7 in cultured D. melanogaster cells. Interestingly, when expressed in mammalian cells DmATP7 does undergo copper-induced trafficking to the plasma membrane and can facilitate copper efflux, demonstrating functional conservation of localisation and trafficking motifs in these P-type ATPases. Malvolio, the orthologue of Divalent Metal ion Transporter 1, also contributes to copper uptake in S2 cells and D. melanogaster, with impaired function associated with sensitivity to copper limitation. D. melanogaster therefore utilises this general metal transporter in addition to the copper-specific Ctr1 pathway. cDNA microarrays were used to identify genes transcriptionally regulated by copper in S2 cells, with RNA interference used to determine whether candidate genes could affect copper homeostasis. Several components of the COPI vesicle trafficking pathway, including ADP-ribosylation factor 1 (Arf1), were found to affect copper levels in S2 cells. Arf1 was found to have a conserved role in regulating copper uptake in cultured mammalian and D. melanogaster cells, and this is likely to be mediated via the localisation of Ctr1 at the plasma membrane. Taken together these studies demonstrate the value of D. melanogaster S2 cells for the study of copper homeostasis, thereby complementing the D. melanogaster model system. These novel findings should stimulate additional research in both D. melanogaster and mammalian systems and facilitate a greater understanding of copper homeostasis.