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Fig. 3. Cholesterol modification is required for membrane association and full
activity of Hh. (A,B) Cl8 cells were treated with Cd to induce
expression of either Hh-Np (top) or Hh-N (bottom). Permeabilised cells (A) or
unpermeabilised cells (B) were subjected to immunostaining for Hh (green) and
for the ER marker BiP (red). Hh-N and Hh-Np are present in large cytoplasmic
vesicles marked with BiP (A). Hh-Np is also present at the plasma membrane
(A). In non-permeabilised cells (B), Hh-N was undetectable, while Hh-Np was
enriched at the plasma membrane as aggregates (arrows). Even without Cd
induction, Hh accretions were faintly detectable because of the leaky
promoter. (C, top) Media of cultured Cl8 cells expressing either Hh-N
or Hh-Np were subjected to gel filtration on a Sephacryl S200 column. Hh-N is
recovered only in the low molecular weight fractions corresponding to the Hh
monomeric form (peak B). Hh-Np was greatly enriched in the high molecular
weight fractions (peak A around 160 kDa). (C, bottom) When subjected to a
Superose 6 column, a stable peak of Hh-Np was recovered around 200 kDa.
Immunoreactivity was also observed in the highest molecular weight fractions.
(D) Peak A- or B-containing fractions were applied to naive Cl8 cells
that were subsequently fixed and stained for Hh. Hh-N was not detectable, but
accretions of Hh-Np were present at the plasma membrane of peak A-treated
cells (arrow). (E) Both peaks were tested for their ability to induce a
Hh reporter gene. At similar concentrations, Hh-Np (peak A) is threefold more
potent than Hh-N (peak B). (F) When peak A is added to naive cells, it
stimulates Fu phosphorylation two to three times faster than peak B (the upper
arrow on each blot indicates the phosphorylated Fu isoform). (G)
Embryonic extracts were subjected to gel filtration on a Superose 6 column: a
stable peak of Hh is visible around 200 kDa, but Hh is also present in the
very high molecular weight fractions. The last fraction falls outside of the
resolution of the column. (C-G) Representative images and blots from three
independent experiments.
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