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. 2006 May 16;103(20):7841-6.
doi: 10.1073/pnas.0600387103. Epub 2006 May 8.

Nuclear trafficking of Drosophila Frizzled-2 during synapse development requires the PDZ protein dGRIP

Bulent Ataman  1 , James Ashley, David Gorczyca, Michael Gorczyca, Dennis Mathew, Carolin Wichmann, Stephan J Sigrist, Vivian Budnik
Affiliations

Nuclear trafficking of Drosophila Frizzled-2 during synapse development requires the PDZ protein dGRIP

Bulent Ataman et al. Proc Natl Acad Sci U S A. .

Abstract

The Wingless pathway plays an essential role during synapse development. Recent studies at Drosophila glutamatergic synapses suggest that Wingless is secreted by motor neuron terminals and binds to postsynaptic Drosophila Frizzled-2 (DFz2) receptors. DFz2 is, in turn, endocytosed and transported to the muscle perinuclear area, where it is cleaved, and the C-terminal fragment is imported into the nucleus, presumably to regulate transcription during synapse growth. Alterations in this pathway interfere with the formation of new synaptic boutons and lead to aberrant synaptic structures. Here, we show that the 7 PDZ protein dGRIP is necessary for the trafficking of DFz2 to the nucleus. dGRIP is localized to Golgi and trafficking vesicles, and dgrip mutants mimic the synaptic phenotypes observed in wg and dfz2 mutants. DFz2 and dGRIP colocalize in trafficking vesicles, and a severe decrease in dGRIP levels prevents the transport of endocytosed DFz2 receptors to the nucleus. Moreover, coimmunoprecipitation experiments in transfected cells and yeast two-hybrid assays suggest that the C terminus of DFz2 interacts directly with the PDZ domains 4 and 5. These results provide a mechanism by which DFz2 is transported from the postsynaptic membrane to the postsynaptic nucleus during synapse formation and implicate dGRIP as an essential molecule in the transport of this signal.

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Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1.
Fig. 1.
Localization of dGRIP at the NMJ. (A) Genomic structure of dgrip showing exons 1–10. Blue, localization of dgripf05600 and the 2 FRT insertions used to generate the null allele; red, region targeted by dGRIP-RNAi (Upper). Protein structure of dGRIP and organization of PDZ domains (Lower). Green, regions used to generate dGRIP antibodies. (B–D and I) dGRIP immunoreactivity at the NMJ and muscles in a preparation stained with antibodies against dGRIP (green) and HRP (red) shown at low (B and C) and high (D and I) magnification in muscles 6 and 7. Arrowheads, punctate dGRIP immunoreactivity at presynaptic boutons; arrows, punctate dGRIP at the postsynaptic muscle area. (E–H) Localization of dGRIP at the presynaptic microtubule bundle, shown in a preparation labeled with antibodies against dGRIP (green), Futsch (red), and HRP (blue). Arrows, large dGRIP muscle spots; arrowhead, punctate dGRIP immunoreactivity at the peribouton area. (J–M) Localization of large dGRIP spots to Golgi bodies shown at low (J) and high (K–M) magnification in a preparation stained with antibodies against dGRIP (green), Tubulin (blue), and Lva (red). Arrow, Lva-positive dGRIP spot; arrowheads, Lva-negative dGRIP puncta. (M Inset) High-magnification view of a GRIP-Lva spot. (N) Association of Lva-positive spots with microtubules shown in a preparation labeled with Lva (blue), Tubulin (red), and Spectrin (green). All are single confocal slices, except for B, C, and J, which are a Z-series projection. [Scale bar, 70 μm (B and C), 17 μm (E–H), 13 μm (J), 8 μm (D, I, and K–N), and 2.5 μm (M Inset).]
Fig. 2.
Fig. 2.
dGRIP immunoreactivity and quantification of NMJ morphology in larvae expressing dGRIP-RNAi. (A–I) dGRIP immunoreactivity at the NMJ in preparations labeled with antibodies against dGRIP (green) and Spectrin (red) (A–C and G–I) or dGRIP (green) and HRP (red) (D–F), in wild-type larvae (A–C) and larvae expressing dGRIP-RNAi in motor neurons (D–F) or muscles (G–I). (J and K) Number of synaptic (J) and ghost (K) boutons at muscles 6 and 7 (A3) in third-instar larvae of various genotypes. (Scale bar, 13 μm.)
Fig. 3.
Fig. 3.
Ghost boutons lack postsynaptic proteins and most active zones. (A–L) Third instar NMJs in wild-type larvae (A, B, and G–I) and in larvae expressing dGRIP-RNAi postsynaptically (C–F and J–L) in preparations labeled with anti-HRP (green) and anti-DLG (red) (A–F), and anti-HRP (blue), anti-GluRIII (red), and nc82 (green) (G–L). Arrows, ghost boutons; asterisks in G–I, uncommon ghost bouton observed in wild type; arrowhead, HRP-labeled process connecting a ghost bouton with the main arbor; asterisks in J–L, ghost bouton containing nc82 immunoreactivity. [Scale bar, 15 μm (A–F) and 12 μm (G–L).]
Fig. 4.
Fig. 4.
Disrupted synaptic structure in larvae expressing dGRIP-RNAi-post. Cross-section through a type I bouton in wild type (A), and a ghost bouton lacking active zones and SSR (B and C) dGRIP-RNAi-post; arrow, active zone. (C) High-magnification view of a ghost bouton membrane (arrowhead), showing its abnormal ruffled appearance. M, muscle; b, bouton. [Scale bar, 1.2 μm (A and B) and 0.3 μm (C).]
Fig. 5.
Fig. 5.
Abnormal trafficking of DFz2-C from the synapse to the nucleus in larvae expressing dGRIP-RNAi-post. (A and B) Muscle nucleus in wild type (A) and in a dGRIP-RNAi-post larva (B) in preparations stained with antibodies against DFz2-C (green), Tubulin (red), and OSA (blue). (C) Number of DFz2-C immunoreactive nuclear spots. (D) External and internalized DFz2 signal intensity at the NMJ. (E) Number of DFz2-N-positive perinuclear vesicles in the internalization assay. (F–Q) External (blue) and internalized (green) DFz2 at the NMJ of wild type (F–K) and dGRIP-RNAi post (L–Q), at 5 (F–H and L–N) and 60 (I–K and O–Q) min after the antibody-binding step in the internalization assay. NMJs are visualized by the anti-HRP staining (red). (R–W) Internalized DFz2 (green) at the perinuclear area in wild-type (R–T), and dGRIP-RNAi-post (U–W) larvae, at 5 (R and U) and 60 (S, T, V, and W) min after the antibody-binding step. (T and W) High-magnification views of a region in S and V. Arrowheads, internalized DFz2 vesicles at the perinuclear area. n, nucleus. [Scale bar, 10 μm (A and B), 20 μm (F–Q), 12 μm (R–V), and 3 μm (T and W).]
Fig. 6.
Fig. 6.
Colocalization of internalized DFz2 with dGRIP and interactions between dGRIP and DFz2. (A and B) Localization of DFz2 (A) and dGRIP (B) at the NMJ. (C–E) Colocalization between dGRIP-RFP (red) and internalized DFz2 (green) at the NMJ. (C′–E′) High-magnification views of the regions enclosed by squares in C–E. (F) Coimmunoprecipitation of V5 (dGRIP) by anti-Myc (DFz2) in S2 cells. (G) Coimmunoprecipitation of dGRIP deletion constructs. Input lanes in F and G correspond to 10% of the initial extract. Molecular masses are shown in kDa. [Scale bar, 10 μm (A–E) and 2 μm (C′–E′).]
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