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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.
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