Wingless signaling at synapses is through cleavage and nuclear import of receptor DFrizzled2

Abstract

Wingless secretion provides pivotal signals during development by activating transcription of target genes. At Drosophila synapses, Wingless is secreted from presynaptic terminals and is required for synaptic growth and differentiation. Wingless binds the seven-pass transmembrane DFrizzled2 receptor, but the ensuing events at synapses are not known. We show that DFrizzled2 is endocytosed from the postsynaptic membrane and transported to the nucleus. The C terminus of DFrizzled2 is cleaved and translocated into the nucleus; the N-terminal region remains just outside the nucleus. Translocation of DFrizzled2-C into the nucleus, but not its cleavage and transport, depends on Wingless signaling. We conclude that, at synapses, Wingless signal transduction occurs through the nuclear localization of DFrizzled2-C for potential transcriptional regulation of synapse development.

Fig. 1

Localization of DFz2-C and DFz2-N to the same NMJs, but to different subcellular compartments, inside the nucleus (DFz2-C) and at the perinuclear region (DFz2-N). (A and B) Wild-type third instar NMJs from muscles 6 and 7 (A3) stained with antibodies against (A) DFz2-C and (B) DFz2-N. (C through J) Representative muscle nuclei at muscle 6 (A3) in preparations double-stained with antibodies against (C through F) DFz2-C (green) and tubulin (red) and (G through J) DFz-N (blue) and tubulin (Tub) (red). Note that (C to F) and (G to J) were obtained from different preparations. (C, D, G, H) show a confocal slice at a focal plane through the nuclear-cytoplasmic boundary (defined by the microtubular array; tangential), and (E, F, I, J) a confocal slice at a focal plane midway through the nucleus (medial). N, nucleus, arrowheads in C point to cytoplasmic DFz2-C. Scale bar, 9 μm in (A and B), and 8 μm in (C to J).

Fig. 2

Localization of DFz2-C to euchromatin and evidence for cleavage of DFz2. (A and B) Colocalization of (A) DFz2-C (green) and PI (red) and (B) DFz2-C and OSA. In (C to E), muscle nuclei were double-labeled with antibodies against the heterochromatin-specific protein HP-1 (red) and DFz2-C (green). Arrows point to regions of adjacent DFz2-C and HP-1 immunoreactivity. Scale bar, 9 μm in (A to C), and 6 μm in (D and E). (F) Western blot of S2 cells transfected with full-length DFz2, DFz2-N, and DFz2-C. (G) Western blots of body wall muscle extracts from wild-type larvae and larvae overexpressing full-length DFz2. (H) Western blot of S2 cells and S2 cells transfected with DFz2 constructs. Blots were sequentially probed with antibodies to DFz2-C, DFz2-N, and tubulin.

Fig. 3

In vivo transport of DFz2 from the cell surface to the nucleus. (A to F) show anti-DFz2-N immunoreactivity at NMJs during the in vivo DFz2 internalization assay. (A and D) Surface DFz2 (blue) and (B and E) internalized DFz2 (magenta) are shown at 5 and 60 min after pulse-labeling. (C and F) show the merged blue and magenta channels. (G and H) DFz2-N immunoreactivity around a muscle nucleus at 5 and 60 min after pulse-labeling. Scale bar, 9 μm in (A to F), and 6 μm in (G and H).

Fig. 4

Role of endocytosis, retrograde transport, and Wg signaling in DFz2-C nuclear import. (A) Mean number of nuclear spots per nucleus in different genotypes. (B and C) Muscle nuclei in larva expressing (B) DFz2 and (C) DFz2 and Shi-DN in the muscles. Tissues were double-labeled with antibodies against tubulin and DFz2-C. Arrow in (B) points to DFz2-C spots that accumulate just outside of the nuclear perimeter. (D) Number of synaptic boutons in wild type and dfz2^C1/Dfdfz2 mutants expressing various DFz2 transgenes in muscle as indicated. ***P < 0.0001 compared with wild type [ANOVA (20)]. (E to G) DFz2-C and tubulin immunoreactivity in (E) S2 cells, and [(F and G)] S2 transfected with DFz2, and treated with and without Wg-conditioned medium. Scale bar, 9 μm in (B) and (C), and 7 μm in (E) to (G).