| Popis: |
Tissue patterning in metazoans is governed by spatially and temporally orchestrated signalling processes. The dorso-ventral patterning of the Drosophila wing by Wingless signalling is one such instance, wherein secreted Wingless from a restricted source interacts with its receptor, DFrizzled2, and results in a distinctive pattern of signalling outputs. This signalling depends on regulated secretion and endocytosis via the conventional Clathrin, Dynamin -dependent pathway (CD pathway). Here, we show that Wingless is mainly internalized through a Clathrin and Dynamin- independent endocytosis pathway with the aid of cell surface GPI-anchored proteins(CG pathway), mediated by Garz, Arf1 and Class I PI3 kinase. These wingless carriers further fuse with endosomes derived from the CD pathway carrying the receptor, DFrizzled2. This culminates in a low pH-dependent transfer of Wingless to its signalling receptor and resulting in effective signalling. The mediation of Wingless signalling by two distinct endocytosis pathways potentially allows cells to autonomously control Wingless uptake independent of the availability of DFrizzled2.To study the localisation and internalisation of Wingless and DFrizzled2, we have standardised uptake assays in live tissues followed by confocal fluorescence microscopy. To further examine the physical interaction of the ligand and its receptor beyond the resolution limit of light microscopy, FRET was used to establish that Wingless binds maximally to DFrizzled2 in the low pH-confined milieu of an endosome rather than at the cell surface where DFrizzled2 competes with other co-receptors to bind Wingless. |
