Wnt-11 controls PAPC membrane localization by blocking its
clathrin-mediated internalization. (A) Cell surface
biotinylation assay performed with DMZ explants, injected with 1 ng
xPAPC-myc RNA, and treated with clathrin- and caveolin1-specific
inhibitors. Cell surface, cytoplasmic (cyto), and total protein amounts
of xPAPC-myc were detected by Western blotting using specific myc tag
antibody (9E10). The relative xPAPC-myc signal intensity (IN)
of cell surface and cytoplasmic fractions were measured as described in
Materials and methods. Finally, the ratio surface versus cytoplasmic
fraction was calculated. The cell surface amount of xPAPC increased in
the presence of clathrin-specific inhibitors. Used clathrin-specific
inhibitors were 60 µM chlorpromazine (CPZ) and 300 µM
monodansylcadaverine (MDC). Used caveolin1-specific inhibitors were 5
µg/ml filipin and 200 µM genistein. neg. KO, negative
control. (B) Confocal microscopic analysis of DMZ explants,
cryosectioned, and immunostained for clathrinHC and xPAPC-EGFP. Nuclei
were stained with DAPI. xWnt-11 expression resulted in clathrin-xPAPC
accumulation at cell membranes. In xWnt-11 morphants, membrane
localization of clathrin and PAPC was strongly reduced but increased in
intracellular vesicles. Injection amount was as follows: 500 pg
xPAPC-EGFP RNA, 20 pg xWnt-11 RNA, and 1 pmol xWnt-11 MO. Bars, 20
µm. (C) Ratio number of membrane versus cytoplasmic
colocalization of xPAPC (xPAPC-clathrin-CL) and clathrinHC in dependency
of xWnt-11 (for more details, see Materials and methods). Error bars
show SEM. Student’s t test was performed
(**, P < 0.005). n, number of
cells.