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First published online February 15, 2008
Journal of Experimental Biology 211, 709-716 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.014159
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Malvolio is a copper transporter in Drosophila melanogaster

Adam Southon1, Ashley Farlow1, Melanie Norgate1, Richard Burke2 and James Camakaris1,*

1 Department of Genetics, The University of Melbourne, Victoria 3010, Australia
2 School of Biological Sciences, Monash University, Victoria 3800, Australia


Figure 1
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  		Fig. 1. Mvl expression and localisation in S2 cells. (A) Western blotting using anti-FLAG antibody detected a 52 kDa band corresponding to Mvl in lysate from pAcMvl cells. No significant signal was visible in lysate from empty vector control cells (pAc). {alpha}-Tubulin was used as a loading control. (B,C) Mvl was detected in S2 cells by immunofluorescence using anti-FLAG and Alexa Fluor 488 anti-mouse antibodies (green) and the nucleus was stained with DAPI (blue). Mvl was detected at the plasma membrane as well as in punctate structures in pAcMvl cells (B), but no significant staining was visible in pAc cells (C).

 

Figure 2
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  		Fig. 2. Cu accumulation in S2 cells. Cu accumulation was measured in S2 cells exposed to 2 µmol l–1 Cu. Intracellular levels were measured after 1 h and 24 h and normalised to total cellular protein. An independent samples t-test was used to determine significant differences. (A) Cu accumulation in S2 cells stably overexpressing Mvl, expressed relative to an empty vector control. Values are mean ± s.e.m. of 15 replicates from four independent experiments. *P<0.0005 compared to control. (B) Cu accumulation following dsRNAi knockdown of Mvl, expressed relative to control. Cells were pre-treated with Mvl or control dsRNA 48 h prior to Cu exposure. Real-time PCR demonstrated Mvl expression was reduced to 18.1±4.6% (mean ± s.e.m.) of control after 48 h. Values are mean ± s.e.m. of 21 replicates from seven independent experiments. {dagger}P<0.0001 compared to control.

 

Figure 3
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  		Fig. 3. S2 cell viability following Cu limitation. S2 cells were pre-treated with Mvl or control dsRNA for 6 days. Real-time PCR demonstrated Mvl was suppressed to 47.3±16.1% (mean ± s.e.m.) of control after 6 days. Cells were exposed to basal medium or Cu-limited medium using 10 µmol l–1 Diamsar for 6 days and viable cells were counted. Values are mean ± s.e.m. of nine replicates from three independent experiments. An independent samples t-test was used to determine significant differences. *P<0.05 compared to basal medium, {dagger}P<0.05 compared to control cells exposed to Cu-limited medium.

 

Figure 4
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  		Fig. 4. Hypopigmentation and thorax development in Drosophila. Hypopigmentation and thorax development were used as markers of Cu deficiency in adult female Drosophila that overexpress ATP7 in the pannier region (boxed in A) using a pnr-Gal4 driver. Overexpression of ATP7 results in a hypopigmentation and a loss of sensory bristles in the pannier region. In addition the width of the thorax is reduced, as indicated by the arrows that represent the distance between the two major posterior sensory bristles. (A) w1118 control flies. (B) Overexpression of ATP7. (C) Overexpression of ATP7 in the Mvl97f/+ background. (D) Co-overexpression of ATP7 and Mvl significantly and reliably further reduced thoracic width and pigmentation.

 

Figure 5
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  		Fig. 5. Drosophila viability following Cu limitation or Cu excess. Larval survival to adulthood was determined for Mvl97f and w1118 Drosophila when raised on Cu-limited medium using 100 µmol l–1 BCS, basal food, or 1 or 2 mmol l–1 Cu. (A) Female survival. (B) Male survival. Values are expressed as percentage emergence and are mean ± s.e.m. of five replicates of 50 larvae. A one-way ANOVA with a Games Howell post-hoc test was used to determine significant differences. *P<0.05 compared to Armenia and {dagger}P<0.05 compared to w1118 control flies.

 

Figure 6
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  		Fig. 6. Larval Cu distribution. Third instar larvae were raised under Cu-limited conditions with 100 µmol l–1 BCS (A,B), basal food (C,D and enlarged in E,F, respectively) or exposed to 1 mmol l–1 Cu for 4 h (G,H) and then dissected. MtnA-EYFP fluorescence was used as a proxy marker of Cu distribution in w1118 control larvae (A,C,E,G) and Mvl97f/+ larvae (B,D,F,H). EYFP levels were not different between male and female larvae (data not shown). Regions of the gut are identified in C: pv, proventriculus; gc, gastric caecum; am, anterior midgut; cc, copper cell region; mm, middle midgut; fe, iron cell region; pm, posterior midgut; mp, Malpighian tubule. Under basal conditions, control larvae (C) showed a complex distribution of EYFP throughout the anterior, middle and posterior midgut as well as the proventriculus and Malpighian tubules. By contrast, Mvl97f/+ larvae (D) did not show the same high levels of EYFP in the proventriculus and anterior midgut (clearly seen in the enlargements E and F). Under Cu-limited conditions, EYFP levels were reduced across all tissues of both control (A) and Mvl97f/+ (B) larvae, with the exception of the iron cell region of the middle midgut and in the imaginal ring at the midgut–hindgut border (indicated by arrows in A). Under Cu-excess conditions EYFP levels were saturated in all tissues of both control (G) and Mvl97f/+ (H) larvae.

 

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