Immunolocalization of the
Arrow protein in Drosophila.
The Wingless protein in
Drosophila signals through a complex of two cell surface receptors: Frizzled2
(Dfz2) and Arrow. Dfz2 is a 7 transmembrane protein with a long aminoterminal
cysteine-rich ligand binding domain (CRD) which contacts Wingless directly.
Arrow is a member of the LRP5/6 family, which are long single pass transmembrane proteins with an
outside EGF repeat domain and an inside proline-rich tail. There is evidence
that Wingless binds to the Arrow EGF repeats and that the intracellular domain
is necessary for signaling. The data on Arrow and Wingless are exciting but very
recent, and there are some controversies. The direct binding of Wg and Arrow
has been disputed, for example.
In intact Drosophila embryos
as well as in imaginal discs, the Wg protein has been detected in cell-internal
vesicles that indicate that the protein is internalized by receptor-mediated
endocytosis. There is also evidence that Wg can be transported over a fairly
long range and can act as a morphogen. The interaction between Wg and its
receptors is probably important in determining the range over which Wg can
travel.
One approach to test whether Wg interacts with Arrow in vivo, or to examine whether the internalization and transport of Wg proceeds through the receptors, is to localize these components by immunostaining. Do the vesicles that stain for Wg also stain for Arrow or Dfz2? Do the two receptors co-localize on the surface of intact cells? Is the staining for one component changed in a mutant for the other component?
In our lab, we have generated
antibodies to several components of Wg signaling, including polyclonal and
monoclonal antibodies to Wg and Dfz2, as well as a recent polyclonal antibody
to Arrow (Michael Povelones). The latter one does detect the Arrow protein in
cultured cells by western blotting and IP, but has yet to be tested in staining
experiments. This antiserum could be an unique and new reagent to test the
current models of wg-receptor interactions.
Initial experiments would be
to affinity-purify the Arrow antibody;
to stain wild type and arrow
mutant Drosophila embryos; and then to co-localize the various components.
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