Fig. 3. Vesicle trafficking defects in sec5^E10. (A) A lateral bd sensory neuron in a wild-type larva that had been fed RU486 at 48 hours AEL and dissected at 59 hours AEL. Anti-HRP immunostaining (gray) was used to find bd sensory neurons in each animal. (B) A lateral bd neuron as in A but from a sec5^E10 mutant fed RU486 at 72 hours AEL and dissected at 83 hours AEL. sec5^E10 larvae show less cell surface CD8 than the control. (C,D) Additional wild-type and sec5^E10 neurons as in A and B. (E) From quantitative fluorescent microscopy of the transport assay, surface-expressed CD8 immunofluorescence, total GFP fluorescence and cell surface area (measured by HRP immunoreactivity) were expressed as fluorescent units after background subtraction. In the sensory neuron soma [n=7 for wild-type larvae fed RU486 (white) and uninduced (blue); n=6 for sec5^E10 larvae fed RU486 (gray) and uninduced (black)], there are comparable levels of anti-HRP labeling at the cell surface. Total GFP fluorescence is reduced in the RU486-fed mutants (gray) compared with RU486-fed controls (white), but there is a significant induction of the GFP signal, which is a measure of transgene expression, in the RU486-fed (gray) compared with uninduced (black) mutants. The amount of mCD8 at the cell surface is reduced in the RU486-fed mutants compared to RU486-fed controls, and a 50% reduction is observed when surface mCD8 is normalized to GFP in order to control for the level of transgene expression (P=0.004). (F) To measure the amount of transgene induction by feeding RU486, the uninduced averages (blue and black bars in E) were subtracted from the RU486-induced averages (white and gray bars in E). To control for differences in cell size, induced GFP and induced surface CD8 were then normalized to the anti-HRP signal. The amount of induced mCD8 trafficked to the cell surface was also normalized to the induced GFP signal, revealing the disruption of transport of the newly synthesized protein to the surface.