Figure 9. Expression of GFP-LegC3-His7X and GFP-His7X in D. discoideum.

(A) D. discoideum cells containing GFP-His7x, or GFP-LegC3-His7x under the control of a Tc-repressible promoter were grown with the presence of 10 µg/mL of Tc for 3 days at 25°C. The cells were washed in SorC buffer and incubated for an additional 8–10 hours in media without tetracycline. Expression of the fusion proteins was visualized under epifluorescence microscopy. (B) D. discoideum cells expressing hybrid fusion proteins were sorted for GFP+ cells after 10 hours of induction. The samples were then fixed and processed for electron microscopy using osmium tetroxide staining. Cells expressing GFP-His7X contain a mixture of vesicles with (1) undigested material, (2) partly digested material and (3) completely digested material. At least half of the cells expressing GFP-LegC3-His7X contain (4) an abnormally high number of vesicles with partly digested contents. What appears to be unfused pro-lysosomes (5) are segregated to a separate portion of the cell. (C) At least 100 cells of each sample were analyzed for abnormal fine structures. The percentage of GFP-LegC3-His7X expressing cells containing abnormal vesicle accumulation versus GFP-His7X expressing cells. 95% confidence intervals are indicated above.