Fig. 1. The large punctate structures of Hh, HhN and Hhc85s are endocytic vesicles and only the lipid-modified Hh forms oligomers in Drosophila. (A) Glycerol gradients (50-15%) used to fractionate Hh-GFP, HhN-GFP, Hhc85s-GFP and Hhc85sN-GFP obtained from salivary glands expressing the four Hh forms. We observed that wild-type Hh fractionated as two different density gradients: one associated with the high molecular weight fraction (9-14), which corresponded to oligomers; the other associated with the low molecular weight fraction (17-23), corresponding to monomers. HhN-GFP, Hhc85s-GFP and Hhc85sN-GFP elute with the low-density fractions (monomers). (B-D,b-d) Wing imaginal discs expressing the different Hh-GFP forms with the hh-Gal4 driver. Hh-GFP forms a short gradient of around six or seven cell diameters (blue line in B), while HhN-GFP and Hhc85s-GFP proteins are unable to generate a proper gradient (blue line in C and D). HhN-GFP and Hhc85s-GFP vesicles are present far from the Hh-expressing cells. Internalized dextran red-positive vesicles (24 minutes of incubation) co-localized with Hh-GFP (b), HhN-GFP (c) and Hhc85s-GFP (d) in both A and P compartments of the wing imaginal cells that expressed the three forms of Hh in their own expression domain using the Hh-Gal4 driver (arrowheads). Some GFP labeled particles do not co-localize with the internalized dextran (arrows). These particles are nevertheless intracellular (see Fig. S1D in the supplementary material). These results indicate that, in all cases, the punctate structures are endocytic vesicles. The A compartment is orientated towards the left; the P compartment is orientated towards the right.