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. 2009 Oct 16;139(2):393-404.
doi: 10.1016/j.cell.2009.07.051.

Trans-synaptic transmission of vesicular Wnt signals through Evi/Wntless

Ceren Korkut  1 , Bulent Ataman, Preethi Ramachandran, James Ashley, Romina Barria, Norberto Gherbesi, Vivian Budnik
Affiliations

Trans-synaptic transmission of vesicular Wnt signals through Evi/Wntless

Ceren Korkut et al. Cell. .

Abstract

Wnts play pivotal roles during development and in the mature nervous system. However, the mechanism by which Wnts traffic between cells has remained elusive. Here we demonstrate a mechanism of Wnt transmission through release of exosome-like vesicles containing the Wnt-binding protein Evenness Interrupted/Wntless/Sprinter (Evi/Wls/Srt). We show that at the Drosophila larval neuromuscular junction (NMJ), presynaptic vesicular release of Evi is required for the secretion of the Wnt, Wingless (Wg). We also show that Evi acts cell-autonomously in the postsynaptic Wnt-receiving cell to target dGRIP, a Wg-receptor-interacting protein, to postsynaptic sites. Upon Evi loss of function, dGRIP is not properly targeted to synaptic sites, interfering with postsynaptic Wnt signal transduction. These findings uncover a previously unknown cellular mechanism by which a secreted Wnt is transported across synapses by Evi-containing vesicles and reveal trafficking functions of Evi in both the Wnt-producing and the Wnt-receiving cells. For a video summary of this article, see the PaperFlick file with the Supplemental Data available online.

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Figures

Figure 1
Figure 1. Wg localization at the NMJ is regulated by presynaptic Evi
(A–D) Single confocal slices of NMJs labeled with anti-HRP and anti-Wg in (A) wild type, (B) an evi mutant expressing transgenic Evi in motorneurons (evi, Evi-pre), (C) an evi mutant, and (D) an evi mutant expressing transgenic Evi in muscles (evi, Evi-post). (E) Normalized Wg levels inside synaptic boutons (pre-) and at the postsynaptic region (post-). (F) Western blot of larval brain extracts. Numbers at the right of the blot represent molecular weigh in KDa. Calibration bar is 7 µm.
Figure 2
Figure 2. Mutations in evi mimic abnormal synaptic phenotypes observed in wg mutants
(A–D) Confocal images of NMJs labeled with anti-HRP and anti-DLG in (A, C) wild type, and (B, D) evi. (A, B) Projections of entire NMJs. (C, D) Single confocal slices of NMJ branches (arrowheads in C, D= abnormal boutons; arrows= ghost boutons). (E–G) Number of (E, G) boutons and (F) ghost boutons. Calibration bar is 30 µm for A, B and 13 µm for C, D.
Figure 3
Figure 3. Evi is localized pre- and postsynaptically at the NMJ and it is transported trans-synaptically as an intact protein
(A) Predicted structure of Evi and the regions (underlined) used for generation of the Evi-Nex and Evi-Cin antibodies. (B–E)Single confocal slices of NMJs double stained with anti-HRP and antibodies to (B, D) Evi-Nex and (C, E) Evi-Cin, in (B, C) wild type, (D, E) evi. Single confocal slices of (F) Evi-RNAi-pre stained with Evi-Nex and HRP, (G) Evi-GFP-pre triple stained with anti-GFP, anti-HRP and anti-Dlg or (H) evi;Evi-GFP-pre stained with anti-GFP, anti-HRP. (I) Normalized pre- and postsynaptic Evi levels. (J) Single confocal slices of a bouton at (J1–J3) low and (J4–J6) high magnification in Evi-GFP-pre stained with anti-GFP, anti-Wg and anti-HRP. (L,M) Images of NMJs from evi;Evi-GFP-pre triple stained with GFP, anti-HRP and antibodies to (L2,L3) Evi-Nex or (M2,M3) Evi-Cin. (N, O) Models on the potential mode of Evi trans-synaptic transfer. In (N) an extracellular region of Evi is cleaved and transported to the postsynaptic compartment. In (O) Evi is transferred as an intact protein through the use of vesicular compartments. Calibration bar is 2 µm for Fig. 3E 5–8 and 6 µm for the rest of the panels.
Figure 4
Figure 4. Evi is transferred from cell to cell and to the medium
(A, B) Single confocal slices of S2 cells (A) either untransfected (outlined by white circles) or transfected with Evi-GFP and (B) either transfected with mCherry or Evi-GFP (arrowheads= Evi-negative cells; arrows= Evi in the media). (C) Evi-GFP and Wg are transferred together into an untransfected cell (arrowheads) (D) Wg localizes with Evi into punctuate structures within filopodia (arrows), as well as in the medium (arrowhead) (E) Western blot of lysates and media from Evi-GFP transfected S2 cells. (F) Time-lapse imaging of an S2 cell transfected with Evi-GFP showing the shedding of an Evi-GFP vesicle to the medium (arrows). Calibration bar is 3µm for panel 4D and 8 µm for the rest of the panels. Time points in 4F are in min.
Figure 5
Figure 5. Evi is localized to pre- and postsynaptic vesicular structures as well as pre- and post-perisynaptic membranes
(A–K) Electron micrographs of synaptic bouton regions in preparations stained with antibodies to Evi-Nex or GFP, labeled with silver intensified 1.4nm gold secondaries, or antibodies to Evi-Cin labeled with 18 nm gold secondaries. The presynaptic compartment has been overlayed in pink, and insets are high magnification views of the structures indicated by the arrows. In (A, B, D–H) samples were stained pre-embedding with anti-Evi-Nex or anti-GFP. In (C) samples were processed for an internalization assay. (I–K) Samples were stained post-embedding with anti-Evi-Cin. (A, B) Immunoreactive vesicles found at the SSR region. (C) Internalized Evi is found in postsynaptic SSR vesicles. (D, K) Localization of label at SSR membranes. (E–H) Evi label at the perisynaptic region of pre- and postsynaptic membranes. Arrowheads in (F) mark the active zone. (I) Evi localization at a presynaptic multimembrane body. (J) Evi-immunoreactive gold particles at the presynaptic region and the synaptic cleft. Calibration bar is 0.6µm in A, C, D, K; 0.3µm in B, E–H; 0.2µm in the insets of A; 0.15µm in the inset of B, and 0.1 µm in the inset of C.
Figure 6
Figure 6. Evi downregulation in muscle results in postsynaptic Wg and DFz2 accumulation and alterations in the Frizzled Nuclear Import Wg pathway
(A–D) Single confocal slices of NMJs triple stained with antibodies to HRP, DLG and (A, B) DFz2 or (C, D) Wg in (A, C) wild type and (B, D) Evi-RNAi-Post. (E) Wg and DFz2 immunoreactivity levels at the postsynaptic region. (F) Intensity of surface and internalized DFz2 at 5 and 60 min after the antibody-binding step in wild type and Evi-RNAi-post. (G–J) Single confocal slices of NMJs subjected to the internalization assay, showing (G1–J1) surface DFz2 and (G2–J2) internalized DFz2 (G, I) at 5 min and (H, J) 60 min after the antibody binding step, in (G, H) wild type, and (I, J) Evi-RNAi-post. (K, L) Confocal slices of muscle nuclei in preparations stained with anti-DFz2-C and Hoechst in (K) wild type and (L) evi mutants. (M) Normalized number of DFz2-C nuclear spots. Calibration bar is 10 µm for panels A-H1-2, I-L; 5 µm for panels A-D3-4.
Figure 7
Figure 7. Downregulating Evi in postsynaptic muscles alters the localization of dGRIP, and proposed function of Evi in the pre- and postsynaptic compartment
(A, B) Single confocal slices of NMJs triple labeled with antibodies to HRP, dGRIP and Lva in (A) wild type, and (B) Evi-RNAi-post (arrows in A= synaptic dGRIP; arrowheads in A, B= Golgi bodies. (C) dGRIP levels at the postsynaptic junctional region, Golgi bodies, and muscle cortex, in wild type and Evi-RNAi-post. (D) Postsynaptic DFz2 levels in wild type, Evi-RNAi-post and Evi-RNAi-post, dGRIP-post. (E) Bouton number in wild type, evi mutants, and evi mutants expressing dGRIP-post. (F) DFz2C spots in wild type, evi mutants, and evi mutants expressing dGRIP-post. (G) Proposed model for Evi function in the pre- and postsynaptic compartment (see text). Calibration bar is 6 µm for A-B1-3, and 3 µm for A-B4-6.
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References

    1. Ataman B, Ashley J, Gorczyca D, Gorczyca M, Mathew D, Wichmann C, Sigrist SJ, Budnik V. Nuclear trafficking of Drosophila Frizzled-2 during synapse development requires the PDZ protein dGRIP. Proc Natl Acad Sci U S A. 2006;103:7841–7846. - PMC - PubMed
    1. Ataman B, Ashley J, Gorczyca M, Ramachandran P, Fouquet W, Sigrist SJ, Budnik V. Rapid activity-dependent modifications in synaptic structure and function require bidirectional wnt signaling. Neuron. 2008;57:705–718. - PMC - PubMed
    1. Baeg GH, Lin X, Khare N, Baumgartner S, Perrimon N. Heparan sulfate proteoglycans are critical for the organization of the extracellular distribution of Wingless. Development. 2001;128:87–94. - PubMed
    1. Banziger C, Soldini D, Schutt C, Zipperlen P, Hausmann G, Basler K. Wntless, a conserved membrane protein dedicated to the secretion of Wnt proteins from signaling cells. Cell. 2006;125:509–522. - PubMed
    1. Bartscherer K, Pelte N, Ingelfinger D, Boutros M. Secretion of Wnt ligands requires Evi, a conserved transmembrane protein. Cell. 2006;125:523–533. - PubMed

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