Fig. 3

Zip71B is localized to the basolateral membrane to import zinc into the tubular cells. a–c Expression pattern and intracellular localization of Zip71B. Zip71B expression (immunofluorescence in red) in the Malpighian tubules of control larvae (a) and Zip71B-RNAi larvae (b) grown on normal food. Arrows denote Malpighian tubules. Scale bars = 100 μm. c Zip71B (red) is localized on plasma membrane and restricted to the basolateral side of tubule cells. DAPI (blue) shows the nucleus. Arrowheads indicate the basolateral membranes, and arrows indicate the apical membranes. Scale bars = 20 μm. Genotypes of the flies used are NP1093-GAL4/+ for control and Zip71B-RNAi/+; NP1093-GAL4/+ for Zip71B-RNAi. d, e Zip71B RNAi, in contrast to ZnT35C RNAi, resulted in zinc reduction in Malpighian tubules. Zinc changes are indicated by alterations of MtnB-EYFP fluorescence; (d) is on normal food and (e) is on zinc food. When reared on normal food, Zip71B-RNAi and Zip71B, ZnT35C-RNAi flies showed no obvious difference in fluorescence signal compared to controls, while ZnT35C-RNAi flies showed dramatically increased MtnB-EYFP fluorescence in Malpighian tubules. When reared on 1 mM zinc food. ZnT35C-RNAi flies showed further increased MtnB-EYFP fluorescence signal in tubules compared to control. In Zip71B-RNAi and Zip71B, ZnT35C-RNAi flies, MtnB-EYFP fluorescence was significantly decreased in tubules. Scale bars = 100 μm. Genotypes of the flies used are MtnB-EYFP/+; NP1093-GAL4/+ for the control, MtnB-EYFP/+; Zip71B-RNAi/+; NP1093-GAL4/+ for Zip71B-RNAi, MtnB-EYFP/+; ZnT35C-RNAi/+; NP1093-GAL4/+ for ZnT35C-RNAi, and MtnB-EYFP/+; Zip71B, ZnT35C-RNAi/+; NP1093-GAL4/+ for the double RNAi flies