Figure 4. Rab14 is recruited to the phagosome and is required for efficient phagolysosome biogenesis.

(A) Rab14 mutant phagosomes show reduced fusion with lysosomes. Hemocytes expressing the lysosomal marker Spin-GFP (hmlGAL4) were bled out and fixed from wildtype and Rab14 mutant larvae after a 20 min pulse with S. aureus conjugated to AF-594 (magenta) followed by a 20 min chase. Hemocytes were evaluated for co-localization between Spinster-GFP and phagosomes as described previously. (B) Percentage of phagosomes showing recruitment of Spin-GFP is plotted for wildtype and Rab14^null (n=4). (C) Rab14 is recruited to the phagosome. Transgenic UAS-Rab14-mRFP flies were generated and the transgene was expressed in hemocytes using cgGal4. Recruitment of Rab14-mRFP (magenta) after different chase times following an initial 20 min pulse with S. aureus-AF488 (green) was examined in wildtype hemocytes (n=3). (D) A representative image and RGB plot for the 20 min pulse + 10min chase time point is shown. The experiments were conducted at least 3 times with 10–12 larvae in each experiment. A total of 70–90 phagosomes were examined for quantification. Error bars indicate SEM. Data was analyzed by two tailed, paired t-test. ** p<0.01 * p<0.05. Scale bar, 5 μm. (E) Rab14 mutants show impaired late endosome to lysosome trafficking. Fat body from wildtype and Rab14 mutant larvae expressing GFP-LAMP (tubulin GAL4) were dissected out and fixed. Rab14 mutant fat body cells accumulate perinuclear GFP-LAMP puncta which indicates a defect in late endosome to lysosome trafficking. A single cell showing increased perinuclear puncta has been circled. The experiments were repeated at least 3 times with 6–7 larvae for each experiment. Scale bar, 20 μm.