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Fig. 1
Photoswitching time-lapse shows phagocytosis block of germination is long-lasting and NADPH oxidase-independent.
(A) CAF2-dTomato-infected fish were treated with DMSO or DPI and scored at 4 hpi for fungal morphotype and internalization. Filamentous growth is only seen extracellularly, and this difference is highly significant (p<0.001) in both DMSO (n = 11) and DPI (n = 12), as measured by Fisher′s exact test. Data are pooled from three independent experiments. (B–E) CAF2-dTomato-infected Tg(mpeg1:GAL4/UAS:Kaede) fish were treated with DMSO (n = 11) or DPI (n = 7) from two hours pre-infection and throughout imaging. At 4 hpi, only macrophages at the infection site in the hindbrain were photoswitched green-to-red by exposure to 405 nm laser light. Photoswitched fish were imaged every two hours for an additional 18 hours. (B) Representative images of macrophage movement during the time-lapse, with a schematic above depicting the movement of macrophages out of the hindbrain and acquisition of yellow color as fresh green-fluorescent Kaede protein is produced. Scale bar = 50 μm. (C) Representative images of DMSO- (left) and DPI-treated (right) fish at 18 hpi, showing filamentous growth in the DPI-treated but not the control fish. Scale bar = 50 μm. (D) Macrophages at the site of infection were enumerated at each time-point. Green and red bars represent green-fluorescent (native Kaede) and red or yellow-fluorescent (photoswitched Kaede). Solid bars represent averages from control fish and lightly shaded bars from DPI-treated fish. Although all the infected, DPI-treated, fish died by 18 hpi, uninfected, DPI-treated, fish do not die due to this treatment alone (data not shown and Fig. 5D). (E) Movement of photoswitched macrophages from the hindbrain was tracked for two classes of Kaede+ macrophages: those that had internalized fungi and those that did not phagocytose fungi. A best-fit line for each population shows that half of those that did not phagocytose fungi have left by approximately 18 hpi, whereas there is no appreciable emigration of macrophages with internalized yeast from the hindbrain in this time. (D–E) Shown are the averages per fish pooled from three independent experiments of at least two fish per group.