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. 2008 May 22;58(4):507-18.
doi: 10.1016/j.neuron.2008.03.007.

Nervous wreck interacts with thickveins and the endocytic machinery to attenuate retrograde BMP signaling during synaptic growth

Kate M O'Connor-Giles  1 , Ling Ling Ho, Barry Ganetzky
Affiliations

Nervous wreck interacts with thickveins and the endocytic machinery to attenuate retrograde BMP signaling during synaptic growth

Kate M O'Connor-Giles et al. Neuron. .

Abstract

Regulation of synaptic growth is fundamental to the formation and plasticity of neural circuits. Here, we demonstrate that Nervous wreck (Nwk), a negative regulator of synaptic growth at Drosophila NMJs, interacts functionally and physically with components of the endocytic machinery, including dynamin and Dap160/intersectin, and negatively regulates retrograde BMP growth signaling through a direct interaction with the BMP receptor, thickveins. Synaptic overgrowth in nwk is sensitive to BMP signaling levels, and loss of Nwk facilitates BMP-induced overgrowth. Conversely, Nwk overexpression suppresses BMP-induced synaptic overgrowth. We observe analogous genetic interactions between dap160 and the BMP pathway, confirming that endocytosis regulates BMP signaling at NMJs. Finally, we demonstrate a correlation between synaptic growth and pMAD levels and show that Nwk regulates these levels. We propose that Nwk functions at the interface of endocytosis and BMP signaling to ensure proper synaptic growth by negatively regulating Tkv to set limits on this positive growth signal.

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Figures

Figure 1
Figure 1. Synaptic overgrowth in nwk is dominantly enhanced by endocytic mutations
(A–H) Confocal images of NMJ 4 labeled with FITC-conjugated anti-HRP. (A) Wild-type NMJs are relatively unbranched and contain few satellite boutons. (B) nwk NMJs are more branched than wild type and have increased numbers of total boutons and satellite boutons. (C–D) Loss of one copy of endo has no effect on total and a small effect on satellite bouton formation in a wild-type background (C), but results in a large increase in both parameters in a nwk background (D). (E–F) Loss of one copy of dap160 does not affect bouton number or satellite bouton formation in a wild-type background (E), but leads to a significant increase in total and satellite bouton number in nwk mutants (F). (G–H) At the semi-restrictive temperature of 25°C, heterozygosity for shi does not affect total or satellite bouton number at wild-type NMJs (G), but in a nwk background, total bouton number and satellite bouton formation are significantly increased (H). (I–J) Quantification of total (I) and satellite (J) bouton number at NMJ 4 in wild type, nwk1/2, endo2730/+, endo2730 nwk2/+ nwk1, dap160Δ1/+, dap160Δ1/+; nwk1/2, shits1/+, and shits1/+; nwk1/2 backgrounds. * p < 0.05, ** p < 0.01, *** p < 0.001. P-values above individual bars refer to comparisons between nwk and the indicated genotype. Arrows in B point to examples of satellite boutons. Scale bar equals 20 µM.
Figure 2
Figure 2. Nwk physically interacts with dynamin and Dap160
(A) A dynamin-PRD-GST-fusion protein precipitates Nwk-RFP from C155GAL4; UASnwk-RFP fly lysates with high affinity, while GST alone does not. (B) GST-Dap160SH3a-d precipitates Nwk-RFP from cellular lysates. (C) Yeast two-hybrid interactions demonstrate a direct association between full-length Nwk and the proline-rich domain (PRD) of dynamin and between Nwk and the SH3 domains of Dap160. Full-length Nwk is fused to the GAL4 DNA-binding domain (prey) and the dynamin PRD and Dap160 SH3 domains are fused to the GAL4 activation domain (bait). After 3 days at 30°C, yeast transformed with either bait or prey alone grow only in the absence of selection, whereas yeast coexpressing both bait and prey fusion proteins exhibit growth on SD medium lacking either histidine or adenine indicating that transcription of reporter genes has been activated. 3AT inhibits His3p activity and increases the stringency of selection. (D) Dap160-SH3c-d, but not Dap160-SH3a-b, GST-fusion protein precipitates Nwk-RFP from cell lysates. A, B and D are probed with rabbit anti-dsRed; input lanes contain lysate equal to 1/10 of the amount used for the pulldown assays.
Figure 3
Figure 3. Synaptic overgrowth in nwk is sensitive to BMP signaling levels
(A–J) Confocal images of NMJ 6/7 (A, C, E, G and I) and NMJ 4 (B, D, F, H and J) labeled with FITC-conjugated anti-HRP. (A, B) wild type. (C, D) nwk1/2 mutants exhibit synaptic overgrowth. (E, F) witA12/B11 mutants have dramatically undergrown synapses. (G,H) witA12 nwk2 double mutants exhibit synaptic undergrowth that is essentially identical to that observed in wit single mutants. (I, J) Loss of one copy of wit dominantly suppresses synaptic overgrowth of nwk (witA12/+ nwk1/2). (K) Quantification of bouton number at NMJ 4 in wild type, nwk1/2, witA12/B11, witA12 nwk2, witA12/+ and witA12 nwk2/+ nwk1 backgrounds. *** p < 0.001. Scale bars equal 20 µM.
Figure 4
Figure 4. Nwk constrains BMP-induced synaptic overgrowth
(A–D) Confocal images of NMJ 4 labeled with FITC-conjugated anti-HRP. (A) wild type. (B) nwk1/2. (C) Neuronal expression of TkvACT in a wild-type background does not result in an increase in satellite bouton number. (D) TkvACT expression in the absence of nwk results in an increase in satellite boutons over that resulting from loss of nwk alone. (E) Quantification of satellite bouton number at NMJ 4 in wild type, nwk1/2, elavGAL4/UAStkvACT and elavGAL4 nwk2/UAStvkACT nwk2 backgrounds. * p < 0.05. Scale bar equals 20 µM.
Figure 5
Figure 5. BMP-induced synaptic overgrowth is suppressed by neuronal overexpression of Nwk
(A–J) Confocal images of NMJ 6/7 (A, C, E, G and I) and NMJ 4 (B, D, F, H and I) labeled with FITC-conjugated anti-HRP. (A, B) wild type. (C, D) C155GAL4; UAStkvACT/UAStkvACT synapses display an increase in bouton number and satellite bouton formation demonstrating that sufficiently high levels of BMP signaling can induce synaptic overgrowth. (E, F) Neuronal overexpression of Nwk suppresses TkvACT-induced synaptic overgrowth. (G, H) Increased bouton number and satellite bouton formation are observed in DadJ1e4 mutants, demonstrating that loss of negative regulation of BMP signaling results in synaptic overgrowth. (I, J) Synaptic overgrowth in DadJ1e4 is suppressed by neuronal overexpression of Nwk. (K, L) Quantification of total (K) and satellite (L) bouton number at NMJ 4 in wild type, C155GAL4; UAStkvACT/UAStkvACT, UAStkvACT/UAStkvACT, C155GAL4; UAStkvACT UASnwk/UAStkvACT, C155GAL4; UASnwk/+, C155GAL4; UAStkvACT/UASmyrRFP/UAStkvACT, DadJ1e4, DadJ1e4/Dad271-68 and C155GAL4; DadJ1e4 UASnwk/DadJ1e4 backgrounds. * p < 0.05, *** p < 0.001. P-values above individual bars refer to comparisons between wild type and the indicated genotype. Scale bars equal 20 µM.
Figure 6
Figure 6. dap160 interacts with BMP signaling pathway components
(A–F) Confocal images of NMJ 4 labeled with FITC-conjugated anti-HRP. (A) Synaptic overgrowth in dap160Δ1/EMS. (B) dap160Δ1; witA12/B11 double mutants exhibit the wit synaptic undergrowth phenotype. (C) Loss of one copy of wit suppresses synaptic overgrowth in dap160 mutants. (D) Reducing the dose of dap160 by 50% facilitates TkvACT-induced synaptic overgrowth. (E–F) Neuronal overexpression of Dap160 suppresses synaptic overgrowth in neurons expressing two copies of TkvACT (E) and lacking Dad (F). (G) Quantification of total bouton number at NMJ 4 in dap160Δ1/EMS, witA12/B11, dap160Δ1/Δ1; witA12/B11, dap160Δ1/Δ1; witA12/+, C155GAL4; UAStkvACT/+, C155GAL4; dap160Δ1/+; UAStkvACT/+, C155GAL4; UAStkvACT/UAStkvACT, C155GAL4; UAStkvACT UASdap160/UAStkvACT, DadJ1e4/J1e4 and C155GAL4; DadJ1e4 UASdap160/DadJ1e4 backgrounds. (G) Quantification of satellite boutons at NMJ 4 in C155GAL4; UAStkvACT/+, C155GAL4; dap160Δ1/+; UAStkvACT/+, C155GAL4; UAStkvACT/UAStkvACT, C155GAL4; UAStkvACT UASdap160/UAStkvACT, DadJ1e4/J1e4 and C155GAL4; DadJ1e4 UASdap160/DadJ1e4. * p < 0.05, ** p < 0.01, *** p < 0.001. Scale bar equals 20 µM.
Figure 7
Figure 7. Nwk physically associates with Tkv
(A) Z-series projection of C155GAL4; UAStkv-GFP boutons co-labeled with antibodies to Nwk. Note that while both proteins exhibit periactive zone localization, both also localize to discrete puncta. (B) Thin (0.1µm) optical cross section of C155GAL4; UAStkv-GFP boutons co-labeled with antibodies to Nwk. Arrows indicate several examples of colocalization of Nwk and Tkv-GFP to membrane-associated puncta. (C) GST-Tkv-IC precipitates Nwk-RFP from C155GAL4; UASnwk-RFP fly lysates with high affinity. Nwk-RFP is detected with rabbit anti-dsRed. We obtained similar results in analogous experiments using lysate from wild-type flies expressing only endogenous Nwk, excluding potential artifacts due to overexpression of Nwk or the RFP tag (data not shown). (D) GST-Nwk precipitates full-length tagged-Tkv from C155GAL4; UAStkv-GFP lysates as detected by rabbit anti-GFP. Input lanes in C and D contain lysate equal to approximately 1/10 of the amount used for the pulldown assays. (E) Yeast two-hybrid interactions demonstrate direct binding of full-length Nwk (prey) and Tkv-IC (bait). After 3 days at 30°C, only yeast coexpressing both bait and prey fusion proteins exhibit growth on SD medium lacking either histidine or adenine.
Figure 8
Figure 8. pMAD levels vary with Nwk expression and correlate with satellite bouton formation
(A–B, D–H) High magnification images of boutons labeled with rabbit anti-pMAD (red) and mouse anti-Dlg (green) in (A) wild type, (B) nwk1/2, (D) endo2730, (E) C155GAL4; UAStkvACT/+, (F) C155GAL4; nwk1/UAStvkACT nwk2, (G) C155GAL4; UAStkvACT/UAStkvACT, and (H) C155GAL4; UAStkvACT UASnwk/UAStkvACT boutons. pMAD levels are increased in endocytic mutants (B,D) and in response to ectopic BMP signaling (E,G). While loss of Nwk enhances (F), overexpression of Nwk suppresses (H) BMP-induced pMAD expression. (C) A high magnification image of nwk boutons labeled with rabbit anti-pMAD (red) and mouse anti-NC82 (green), which labels the active zone protein Bruchpilot (Brp), shows localization of pMAD to presynaptic terminals. Note that in (B), pMAD does not colocalize with the postsynaptic marker Dlg. (I) Quantification of pMAD levels normalized for control (Discs large) intensity (red bars). pMAD levels show a strong correspondence to satellite bouton number (grey bars). * p < 0.05, ** p < 0.01, *** p < 0.001. P-values above individual bars refer to comparisons between wild type and the indicated genotype. Scale bar equals 5 µM.
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References

    1. Aberle H, Haghighi AP, Fetter RD, McCabe BD, Magalhaes TR, Goodman CS. wishful thinking encodes a BMP type II receptor that regulates synaptic growth in Drosophila. Neuron. 2002;33:545–558. - PubMed
    1. Allan DW, St Pierre SE, Miguel-Aliaga I, Thor S. Specification of neuropeptide cell identity by the integration of retrograde BMP signaling and a combinatorial transcription factor code. Cell. 2003;113:73–86. - PubMed
    1. Bokel C, Schwabedissen A, Entchev E, Renaud O, Gonzalez-Gaitan M. Sara endosomes and the maintenance of Dpp signaling levels across mitosis. Science (New York, N.Y. 2006;314:1135–1139. - PubMed
    1. Budnik V, Zhong Y, Wu CF. Morphological plasticity of motor axons in Drosophila mutants with altered excitability. J Neurosci. 1990;10:3754–3768. - PMC - PubMed
    1. Chen Y, Riese MJ, Killinger MA, Hoffmann FM. A genetic screen for modifiers of Drosophila decapentaplegic signaling identifies mutations in punt, Mothers against dpp and the BMP-7 homologue, 60A. Development (Cambridge, England) 1998;125:1759–1768. - PubMed

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