(A) Schematic of Nwk domain structure and representative confocal images of α-Cpx-stained third instar larval NMJ morphology on muscle 4 for the indicated genotypes. Arrows indicate satellite boutons. (B) Quantification of synaptic growth on muscle 6/7 and satellite bouton number on muscle 4 by Nwk variants in a nwk¹/nwk² null background. Graphs show mean +/− s.e.m. Numbers in bar graphs represent the number of NMJs. (C) Localization of Nwk variants. GFP-tagged Nwk variants were expressed in the nwk¹/nwk² null background, under control of the GAL4 driver elav^C155 (pan-neuronal and salivary glands). Third instar larvae were fixed and stained with α-BRP and α-HRP antibodies. Scale bars are 10 µm. Associated with .

(A) Schematic of Nwk constructs and summary of protrusion formation in cells. (B,C) Activity of GFP-tagged Nwk truncations in S2 cells. Images show GFP fluorescence (in inverted contrast) of a maximum intensity Z-projection. Scale bars are 10 µm. (D) Quantification of cellular morphology (perimeter (P)/square root of area (A)). Data are represented as mean +/− s.e.m. from at least 9 cells per condition, ***p<0.001.

GST fusion proteins were immobilized on glutathione agarose and incubated with the indicated purified proteins. Pellets and supernatants were fractionated by SDS-PAGE, immunoblotted or Coomassie stained, and quantified by densitometry: all graphs show the average +/− s.e.m. of three independent reactions. *p<0.05, **p<0.01, ***p<0.001. (A) Salt sensitivity of 6xHis-Xpress-Nwk^1–428 (1.5 µM) co-sedimentation with GST-Nwk-SH3 domains (2–3 µM). Image shows representative anti-Xpress tag immunoblot of co-sedimentation assay. (B) Phyre model of Nwk SH3 domain (based on BIN1/AMPH2 SH3 (PDB C1mv3A)). (C) Co-sedimentation of Nwk^1–428 (1.5 µM) with GST or GST-Nwk-SH3b variants (3 µM). Image shows representative Coomassie-stained gel. (D) Activity of Nwk^{1–731(E654R)} in S2 cells Image shows GFP fluorescence (in inverted contrast) for a maximum intensity Z-projection. Scale bar is 10 µm. Quantification of cellular morphology (perimeter(P)/square root of area(A)) is shown below. (E) Co-sedimentation of Nwk^1–428 (1.5 µM) with GST-Nwk-SH3b and GST-Nwk-SH3a (3–3.5 µM) depends on the charged dimer tips. Image shows a representative immunoblot. Associated with .

(A) Nwk constructs used for in vitro assays. (B, C) Purified proteins (10 µM Nwk^SH3ab (Nwk residues 536–731); 1 µM all other proteins) were incubated with liposomes of the following composition: 80-X% PC, 15% PE, 5% PS and X% PI(4,5)P₂ (where X is the concentration indicated in the graph) and subjected to liposome cosedimentation assays. Graphs show mean densitometry from one (B) or three (mean +/− s.e.m.) (C) independent experiments. (C) Image shows representative Coomassie staining of supernatant (S) and pellet (P) fractions at 10% PI(4,5)P₂. (D) Cryo-EM of control and Nwk deformed liposomes. Both purified Nwk^1–428 and Nwk^1–731 induce membrane scalloping, pointing, and pinching of 10% PI(4,5)P₂ liposomes. Scale bar is 100 nm. Bar graph summarizes vesicle morphology after 30-minute incubation of Nwk^1–428 or Nwk^1–731 (2µM and 500nM) with 0.3 mM [DOPC:DOPE:DOPS:PI(4,5)P₂] = 70:15:5:10 liposomes. n represents the number of liposomes examined; Associated with .

NBD-PE-labeled GUVs incubated with SNAP-549-tagged Nwk variants (500 nM) were imaged by spinning disk confocal microscopy. Lipid composition was [DOPC:POPE:DOPS:PI(4,5)P₂:NBD-PE] = 75:14.5:5:5:.5. n represents the number of vesicles examined. (A) Percent of protein-decorated GUVs after 30-minute incubation with Nwk^1–428 (blue) or Nwk^1–731 (red). Graph represents mean +/− s.e.m. from 3 independent experiments. Data are identical to 5% PI(4,5)P₂ data point in . (B) Quantification of the morphology of protein-decorated GUVs after 30-minute incubation with purified Nwk^1–428 or Nwk^1–731. GUVs were imaged from n independent reactions. (C) Single spinning disc confocal slices of GUVs showing Nwk^1–731 displaying limited recovery on deformed membranes, while partial recovery is observed on spherical, undeformed Nwk^1–428-coated GUVs. Scale bar is 10 µm. Associated with –. (D) Quantification of recovery of protein fluorescence for Nwk^1–428 and Nwk^1–731. The data are a mean of at least 10 independent experiments and the error bars indicate ± s.e.m. (E) Quantification of protein fluorescence recovery according to vesicle morphology. One-way ANOVA *p<0.05,**p<.005,***p<.001.

Scale bars are 10 µM. (A) Representative images of NBD-PE-labeled GUVs of varying PI(4,5)P₂ concentrations incubated with 500nM SNAP-549-Nwk^1–731 for 30 minutes and imaged by spinning disk confocal microscopy. (B) Percent of vesicles bound by Nwk^1–428 and Nwk^1–731 at various PI(4,5)P₂ concentrations (solid line) and percent of bound vesicles that are deformed (dotted line). Graph represents mean +/− s.e.m. from 2–3 independent experiments. 5% PI(4,5)P₂ data is identical to . (C) A plot of percent bound vesicles vs. percent-deformed (from data in (B)) shows an inverse relationship between the number bound and frequency of deformation (D) Representative images of cell-sized water droplets with 1 µM SNAP549-labeled BAR proteins. Emulsions were made with 23 mM lipid mixes of DPHPC +/− PI(4,5)P₂ in decane and incubated for 1 hour before imaging. Associated with –. (E) Quantification of the percentage of total vesicles with protein-induced deformation at 2.5% and 10% PI(4,5)P₂ for Nwk^1–428 and Nwk^1–731. Number above bars represents the number of droplets examined. Associated with . (F) Comparison of the orientation of Nwk^1–428 dimers on lipid monolayers with 2.5% or 10% PI(4,5)P₂. Error bars represent fraction of particles +/− s.e.m. from 3–4 independent EM grids. Shown below are superimposed 10° rotations of the predicted Nwk^1–313 structure (), and representative class averages from 10% PI(4,5)P₂, grid A, . R represents correlation coefficients to the 20 Å-filtered predicted structure. Associated with .

(A,D) Increasing cellular PI(4,5)P₂ levels with the OCRL inhibitor YU1422670 alters Nwk-induced deformation. Images show GFP fluorescence (in inverted contrast) of maximum intensity z-projections. Scale bar is 10 µm. (B) Nwk^1–731-EGFP expressing cells exhibit a significantly higher protrusion index (P/√A; mean +/− s.e.m.; number in bar graph represents n cells) at low concentrations of YU1422670 compared to DMSO control-treated cells, (C) with a higher percentage of cells with a protrusion index of >5. (E) Nwk^1–428-EGFP expressing cells have a significantly lower protrusion index (P/√A; mean +/− s.e.m.; number in bar graph represents n cells) at high concentration (30µM) of YU1422670 compared to DMSO and 10µM YU1422670, (F) and a lower percentage of cells with a protrusion index of 10 or higher after drug treatment. Associated with