SNX16 physically interacts with Nwk. (A) Schematic of Nwk and SNX16 domain organization and summary of yeast two-hybrid interactions. (B) Alignment of SNX16 homologues between aa 346 and 363 of Drosophila SNX16. NCBI Protein database accession numbers and a full SNX16 alignment are shown in Fig. S1. Shaded glutamate residues are required for Nwk–Snx16 interactions. (C) GST and GST-SNX16 (aa 335–407; coiled coil [CC]) were used to precipitate binding partners from wild-type Drosophila head extracts. Equal amounts of precipitates were immunoblotted with α-Nwk antibodies. The first lane represents 2.5% of the input compared with experimental samples. (D) GST and GST-Nwk (aa 1–731) were used to precipitate binding partners from wild-type, UAS-Snx16, and UAS-Snx16-GFP–expressing Drosophila head extracts. Equal amounts of precipitates were immunoblotted with α-GFP, α-SNX16, or α-Dap160 antibodies. Dap160 has previously been shown to interact with Nwk (O’Connor-Giles et al., 2008; Rodal et al., 2008). The first and fourth lanes represent 2% of the input compared with experimental samples. (E) GST-Nwk (aa 1–731) was coexpressed in E. coli with empty vector, SNX16 (aa 220–407; PX and coiled-coil domains), or SNX16^3A (aa 220–407), precipitated on glutathione agarose, and immunoblotted with α-SNX16 antibodies. A. gambiae, Anopheles gambiae; D. rerio, Danio rerio; R. norvegicus, Rattus norvegicus.

SNX16 localizes to Rab5-positive early endosomes. (A) Kc167 cells transfected with Snx16-GFP were treated with wortmannin and then fixed and imaged by confocal microscopy. Single confocal sections are shown. (B) S2 cells transfected with the indicated constructs were spread on concanavalin A, fixed, and imaged. 2D projections of confocal stacks are shown. (C) Kc167 cells transfected with human transferrin receptor and the indicated constructs were incubated with Alexa Fluor 546–labeled transferrin for the indicated times and then fixed and imaged. Single confocal sections are shown. (D) Nwk localizes to a subset of SNX16 puncta. S2 cells were transfected with Snx16-mRFP and nwk-GFP and spread on concanavalin A before fixation and imaging. A 2D projection of a confocal stack is shown. Arrows indicate overlapping puncta. Bars, 10 µm.

Localization of SNX16 to endosomes at the Drosophila NMJ. (A) elav^C155;UAS-Snx16;UAS-GFP-Rab5 larvae stained with α-SNX16 antibodies. (B) elav^C155;UAS-Snx16-GFP;UAS-mRFP-Rab4 larvae. (C) elav^C155;UAS-Snx16-GFP larvae stained with α-Rab11 antibodies. (D) elav^C155;UAS-Snx16;UAS-tkv-GFP larvae stained with α-SNX16 antibodies. (E) elav^C155;UAS-Snx16-GFP larvae stained with α-Hrs antibodies (also available in the JCB DataViewer). (F) elav^C155;UAS-Snx16-GFP and UAS-Snx16^3A-GFP larvae stained with α-GFP antibodies and processed for immunoelectron microscopy. Bars: (A–E) 5 µm; (F) 100 nm.

Nwk in live NMJs localizes to puncta that transiently interact with SNX16 endosomes. (A) elav^C155;Nwk-mRFP expression in α-Nwk IBs of Drosophila larval fillet extracts. The single asterisk indicates endogenous Nwk; the double asterisk indicates Nwk-mRFP. Molecular masses are given in kilodaltons. tub, tubulin. (B) Localization of Nwk-mRFP in live third instar NMJs. Arrows indicate Nwk-mRFP puncta. (C) Localization of Nwk-mRFP in fixed third instar NMJs. Nwk collapses to discrete structures upon fixation, and Nwk puncta are not strongly detected by α-Nwk antibodies. (D) Nwk-mRFP puncta colocalize with GFP-Rab11 at NMJs. (E) Localization of Nwk-mRFP and SNX16-GFP in live third instar larval NMJs. Time-lapse recordings of these NMJs are shown in Videos 1, 2, and 3. The white box indicates the area shown in the time-lapse images below. Arrows track a pair of Nwk-mRFP and SNX16-GFP puncta. (F) The dynamin inhibitor dynasore drives association of Nwk and SNX16 particles. The quantification shows the mean fraction of Nwk particles with a SNX16 particle within 1 µm of their center. Error bars are means ± SEM, and the number of samples averaged in each measurement is indicated at the base of each bar. **, P < 0.01. Bars, 10 µm.

A Nwk-binding mutant of Snx16 recapitulates the nwk phenotype. (A) Representative confocal projections of anti-complexin (Cpx) staining from NMJs of the indicated genotype under the control of the elav^C155 GAL4 driver. (B) Quantification of the mean bouton number for muscle 6/7. (C) Quantification of the mean bouton number for muscle 4. (D) Quantification of the mean satellite bouton number for muscle 4. (E) Cooverexpression of Nwk-mRFP suppresses the Snx16 but not the Snx16^3A overexpression phenotype. Black bars indicate control animals expressing mRFP, and red bars indicate animals expressing Nwk-mRFP. (F) Snx16^3A overexpression increases BMP signaling. Trio protein levels are increased in muscle 6/7 NMJs. Error bars are means ± SEM, and the number of samples averaged in each measurement is indicated at the base of each bar. *, P < 0.05; ***, P < 0.005. Bars: (A) 20 µm; (F) 5 µm.

Loss of SNX16 suppresses the nwk mutant phenotype. (A) Schematic of snx16 loss-of-function mutants. (B) Quantitative RT-PCR of Snx16 and CG10934 transcript levels. n = 6 for each sample. (C) Quantification of the mean satellite bouton number for muscle 4 NMJs. (D) Quantification of the mean bouton number for muscle 6/7 NMJs. (E) Elevated BMP signaling in nwk NMJs is suppressed in Snx16;nwk NMJs. Error bars are means ± SEM, and the number of samples averaged in each measurement is indicated at the base of each bar. *, P < 0.05; **, P < 0.01; ***, P < 0.005. Def, deficiency. Bars, 5 µm.

Snx16 is required for synaptic growth mediated by BMP and Wg cascades. (A) Quantification of the mean bouton number for muscle 6/7 NMJs from larvae defective in BMP signaling. (B) Quantification of the mean satellite bouton number for muscle 4 NMJs defective in Wg signaling. (C) Representative confocal images of muscle 4 NMJs from Snx16-GFP;tkv^Q199D larvae and quantification of the mean satellite bouton number. Control UAS data are identical to Fig. 5 E. (D) Localization of Tkv^Q199D and endogenous Tkv to Snx16-positive endosomes. Quantification shows muscle 4, segments A2–A4, α-Tkv–integrated density in GFP-positive pixels relative to total NMJ α-Tkv–integrated density in single confocal sections. Error bars are means ± SEM, and the number of samples averaged in each measurement is indicated at the base of each bar. **, P < 0.01; ***, P < 0.005. Bars: (C) 20 µm; (D) 10 µm.

A branched pathway model for the attenuation of synaptic growth signals by endosomal traffic. (A) Model depicting how Nwk and SNX16 may drive tubulation-based sorting of synaptic growth receptors from the signal-permissive early endosome to the recycling endosome, where signaling (indicated by yellow starbursts) is attenuated. In the absence of Nwk or Nwk–SNX16 interactions, receptors are retained in the early endosome. In the absence of SNX16, receptors are down-regulated by internalization into the endosomal lumen. (B) Representative confocal images of Cpx-stained NMJs from hrs^D28/Df(exel6277) and Snx16^PB, hrs^D28/Snx16^Δ1, Df(exel6277) larvae and quantification of the mean bouton number. (C) Overexpression of SNX16^3A increases MVB number. Electron micrographs of type 1b boutons from SNX16-GFP and SNX16^3A-GFP–expressing larvae and quantification of MVB structures. Error bars are means ± SEM, and the number of samples averaged in each measurement is indicated at the base of each bar. *, P < 0.05; **, P < 0.01; ***, P < 0.005. Bars: (B) 10 µm; (C) 200 nm.