(A) Electroretinograms of controls (yw eyFLP; P{y⁺} FRT40A / l(2)cl-2L P{w⁺} FRT40A), tweek mutants (yw eyFLP; tweek^{1 or 2} P{y⁺} FRT40A / l(2)cl-2L P{w⁺} FRT40A) and rescued tweek animals (yw eyFLP; tweek ² P{y⁺} FRT40A; tweek⁺(HB69) / +). The positions of ‘on’ and ‘off’ transients (or lack thereof) are indicated by grey arrows.
(B–C) Electron microscopy of control (yw eyFLP; P{y⁺} FRT40A / l(2)cl-2L P{w⁺} FRT40A) and tweek¹ mutant (yw eyFLP; tweek¹ P{y⁺} FRT40A / l(2)cl-2L P{w⁺} FRT40A) lamina cartridges. PR terminals of one cartridge are artificially labeled in green.
(D–E) Electron microscopy images of single PR terminals of control (C) and tweek¹ mutant (D) animals. Capitate projections (arrowhead) and mitochondria (m) are indicated.

(A–D) FM 1–43 dye uptake experiment on synj¹/+x controls (yw ey-FLP; FRT42D synj¹ / FRT42D), tweek²/Df mutants (yw eyFLP; tweek² P{y⁺} FRT40A / Df(2L)Exel8036) and tweek²/Df mutants that lack one functional copy of the synj gene (yw ey-FLP / yw; tweek ² synj¹ / Df(2L)Exel8036). Preparations were stimulated in 90 mM KCl for 5 min, washed and imaged (A–C) and labeling intensity quantified (D). Dye uptake in synj1/+ and yw eyFLP; FRT40A controls is indistinguishable (shown in D). Note the increased FM 1–43 dye uptake in tweek mutants with reduced synj function compared to tweek mutants. To measure PI(4,5)P₂ levels in synj¹ and synj¹/+ animals we expressed the PLCδ-PH-EGFP probe and measured boutonic fluorescence relative to controls: 100±12% for controls (elav-GAL4/Y; P{w⁺UAS-PLCδ-PH-EGFP} / +); 157±20% p<0.05 for synj¹ (elav-GAL4/Y; P{neoFRT}42D synj¹; P{w⁺UAS-PLCδ-PH-EGFP} / +); 117±8% p<0.1 for synj¹/+ (elav-GAL4/Y; P{neoFRT}42D synj¹/+; P{w⁺UAS-PLCδ-PH-EGFP} / +).
(E–H) α-adaptin labeling in synj¹/+ controls, tweek²/Df mutants and tweek²/Df mutants that lack one functional copy of the synj gene (tweek² synj¹/Df). (H) Quantification of α-adaptin labeling intensity. α-adaptin labeling in synj¹/+ and yw eyFLP; FRT40A controls is indistinguishable (shown in D). Note the increased α-adaptin labeling in tweek mutants with reduced synj function compared to tweek mutants. * p<0.05, ** p<0.01 (t-test), Error bars: SEM, n (the number on animals labeled) is indicated in the bars.

(A–F) Ultrastructure of control (yw; P{y⁺} FRT40A) (A, E) and tweek²/Df (yw eyFLP; tweek² P{y⁺} FRT40A / Df(2L)Exel8036) mutants (B–D, F) NMJ boutons (A–D) and dense bodies (E–F). Note the reduced synaptic vesicle density and the heterogeneity in synaptic vesicle size (asterisks) in the mutants compared to the control. Dense bodies (arrowheads) and mitochondria (m) are marked. Scale bars are 200 nm.
(G–K) Quantification of ultrastructural features: mitochondrial density (G), boutonic area per dense body (H), dense bodies per perimeter bouton (I), number of docked vesicles synaptic in a 200 nm radius around the dense body (J) and synaptic vesicle density (K). ** p<0.01 (t-test), ns, not significant; Error bars: SEM, The number of analyzed sections is indicated in the bars and images were acquired from 8 boutons in 5 different animals.
(L–M) Histograms of synaptic vesicle diameter in controls and tweek²/ Df and cumulative histogram of synaptic vesicle diameters indicating larger vesicles in tweek mutants. 737 vesicle diameters were measured for each genotype.

(A–F) Confocal images showing labeling of control (yw; P{y⁺} FRT40A) (left) and tweek²/Df (yw eyFLP; tweek² P{y⁺} FRT40A / Df(2L)Exel8036) (light) larval filets with α-adaptin (A), AP180/Lap (B), StonedB (C), Dynamin (D), Dap160/Intersectin (E) Endophilin (F) (green) and DLG/PSD-95 (magenta). Green channel labeling for control and tweek²/Df is shown in the middle (gray scale).
(G–H) Quantification of boutonic labeling intensity (inside the respective DLG circumscribed areas) for markers shown in (A–F) and for Eps15. Data for tweek¹/tweek² mutants (yw eyFLP; tweek¹ P{y⁺} FRT40A / tweek² P{y⁺} FRT40A) is very similar to tweek²/Df mutants (not shown). * p<0.05, ** p<0.01 (t-test), Error bars: SEM, n (the number on animals labeled) is indicated in the bars.
(I) Western Blots of larval extracts of controls, tweek¹/tweek² and tweek²/Df using antibodies against the endocytic proteins tested in (A–H). Protein loading amounts were tested with anti-actin antibodies. Quantification of 3 independent Westerns normalized to actin loading control (values relative to control levels): α-adaptin: control: 100.0±12.7%; tweek¹/tweek²: 95.8±19.1%; tweek²/Df(2L)Exel8036: 86.4±20.3%; p:ns. Lap/AP180: control: 100.0±6.4%; tweek¹/tweek²: 94.2±8.9%; tweek²/Df(2L)Exel8036: 82.3±21.2%; p:ns. stonedB: control: 100.0±23.0%; tweek¹/tweek²: 87.4±15.3%; tweek2/Df(2L)Exel8036: 81.5±2.4%; p:ns.

(A) Cumulative histogram of mEJPs measured from controls (black: yw; P{y⁺} FRT40A) and tweek¹/tweek² (red: yw eyFLP; tweek¹ P{y⁺} FRT40A / tweek¹ P{y⁺} FRT40A) animals. Note the rightward shift in tweek mutants signifying larger mEJP amplitudes.
(B–C) Average mEJC amplitude (B) and frequency (C) in controls and tweek mutants (tweek²/Df :yw eyFLP; tweek² P{y⁺} FRT40A / Df(2L)Exel8036 and tweek^c01084/ tweek²: yw eyFLP; tweek² P{y⁺} FRT40A / tweek^c01084).
(D) Average EJC amplitude in controls and tweek mutants recorded in 1 mM extracellular Ca²⁺.
(E–F) Sample EJC (E) and mEJC (F) traces recorded from controls and tweek mutants. Recordings were performed for 1 min at 0.1 Hz and all EJC amplitudes were averaged per recording.
(G) Junctional quantal content at 1 mM Ca²⁺ calculated by dividing the average EJC amplitude by the average mEJC amplitude. ** p<0.01 (t-test), Error bars: SEM, n (the number of animals tested) is indicated in the bars.
(H) Estimation of vesicular membrane added per stimulus in 1 mM extracellular Ca²⁺ calculated by multiplying the quantal content (control: 189 quanta; tweek²/ Df: 120 quanta) by the average vesicle surface area based on TEM in Figure 4 (control: vesicle radius 16.7 nm; tweek²/ Df: vesicle radius 23.8 nm).

(A) Average EJP amplitude recorded in 1 mM Ca²⁺ in controls, tweek¹/tweek², tweek₂/Df and tweek^c01084/tweek². Recordings were performed for 1 min at 1 Hz and 60 EJP amplitudes were averaged per recording. Under these conditions, there are no exocytic defects in tweek mutants.
(B) Average EJP amplitudes recorded at 10 Hz for 10 min in 5 mM external Ca²⁺ in controls, tweek¹/Df, tweek²/Df, tweek^c01084/tweek² and tweek¹/tweek²; tweek⁺(+HB69) rescued animals. Average EJP amplitudes (binned per 30 s) are normalized to the initial response (an average of the first 5 EJPs).
(C–F) FM 1–43 dye uptake in controls (yw; P{y⁺} FRT40A) (C,D) or (UAS-DCR2 / w¹¹¹⁸; nSyb-Gal4 / ⁺) (E,F), tweek¹/tweek² mutants (yw eyFLP; tweek¹ P{y⁺} FRT40A / tweek² P{y⁺} FRT40A), tweek²/Df mutants (eyFLP; tweek² P{y⁺} FRT40A / Df(2L)Exel8036), tweek^c01084 / tweek² mutants (eyFLP; tweek² P{y⁺} FRT40A / tweek^c01084), tweek¹/tweek² mutants with a rescue construct (yw eyFLP; tweek¹ P{y⁺} FRT40A / tweek² P{y⁺} FRT40A; tweek⁺(HB69)/+) (C,D) and flies that express RNAi directed against TWEEK (UAS-DCR2 / w¹¹¹⁸; 8060GD / +; nSyb-Gal4 / + or UAS-DCR2 / w¹¹¹⁸; nSyb-Gal4 / 19305GD or 19306GD) (E,F). Preparations were incubated in 4 µM dye and were stimulated with 1 min of 90 mM KCl to label the exo-endo cycling pool.
* p<0.05; ** p<0.01 (t-test), ns: not significant, Error bars: SEM, n (the number of animals tested) is indicated in the bars.

(A–C) Neuronal PI(4,5)P₂ levels in boutons of control (elav-GAL4/Y; UAS-PLCδ-PH-EGFP / +), synj¹ (elav-GAL4/Y; FRT42D synj¹; UAS-PLCδ-PH-EGFP / +) and tweek¹/tweek² (elav-GAL4/Y; tweek¹ P{y⁺} FRT40A / tweek² P{y⁺} FRT40A; UAS-PLCδ-PH-EGFP / +) third instar filets were visualized by a PLCδ-PH-EGFP probe (green). Neuronal membranes were counterstained with anti-HRP (magenta). Green channel is separately shown on the bottom. Note increased EGFP levels in synj1 mutants and decreased EGFP levels in tweek mutants.
(D) Quantification of PLCδ-PH-EGFP intensity shown in (A–C) inside the volume demarcated by anti-HRP labeling in the indicated genotypes. * p<0.05 (t-test)
(E–H) Live imaging of PLCδ-PH-EGFP localization before (E, F, G and H) and after 40s (G’, H’) or 100s (G”, H”) of 20 Hz stimulation of (E and G–G”) control (yw /w; FRT40A /+; UAS-PLCδ-PH-EGFP, nsyb-Gal4/+) and (F and H–H”) tweek²/Df (yw /w; tweek² FRT40A / Df(2L)Exel8036; UAS-PLCδ-PH-EGFP, nsyb-Gal4/+) third instar NMJs. GFP imaging was performed with a CCD camera and G and H are magnifications of E and F respectively. Controls never show PLCδ-PH-EGFP clusters (n= 7 animals) whereas numerous PLCδ-PH-EGFP clusters (green and blue arrowhead) appear in tweek mutants during stimulation. Such clusters are not observed using the PLCδ-PH^MUT-EGFP, indicating specificity. Scale bar: 5 µm.
(I–J) Quantification of the fluorescence of PLCδ-PH-EGFP clusters during stimulation (start at t=0, marked by the bar). (I) Green and blue traces show the normalized fluorescence of individual clusters indicated in H–H”, showing that some clusters form early in the stimulus (green) and others form later (blue). Note also that clusters remain for an extended period of time (>100 s) following stimulation before they dissapear (J) Black trace shows the average ± SEM for all clusters formed in the tweek²/Df experiments (23 clusters, 4 animals). The red trace shows the corresponding change in fluorescence in the remainder of the terminal of tweek²/Df, and this trace appears very similar to the one measured from control boutons where also no clusters were observed (G–G”).
(K–L) Neuronal PI(3)P levels in boutons of control (elav-GAL4/Y; UAS-2xFYVE-EGFP / +) or tweek¹/tweek² mutants (elav-GAL4/Y; tweek² UAS-2xFYVE-EGFP / tweek¹ P{y⁺} FRT40A) third instar filets were visualized with a 2xFYVE-EGFP probe (green). Neuronal membranes were counterstained with anti-HRP (magenta). Green channel is separately shown on the bottom.
(M–N) Neuronal GFP levels in boutons of control (elav-GAL4/Y; UAS-GFP / +) or tweek¹/tweek² mutant (elav-GAL4/Y; tweek² UAS-GFP / tweek¹ P{y⁺} FRT40A) third instar filets (green). Neuronal membranes were counterstained with anti-HRP (magenta). Green channel is separately shown on the bottom.

(A) P-element mapping. P-elements used for mapping: numbers separated by a “/” indicate the number of recombinants out of flies scored. Recombination distance in cM for two nearby P-elements is indicated. The cytological interval and the Exelixis deficiencies that complement (Green) or not (Red) are shown. The area magnified in (B) is shown by a grey arrow
(B) The mapping location of tweek (Blue) based on recombination data (Star). CG15133, CG15134 and CG4841 correspond to the tweek gene. EY02528, as well as c01084 fail to complement the tweek^{1 and 2} alleles. The regions cloned in P[acman] to create rescue constructs are indicated: red constructs do not rescue the tweek alleles while the green constructs do rescue the tweek alleles.
(C) Intron-exon structure of tweek and RT-PCR analysis. Start codons are marked in green and stop codons in red. The P-element excision EY02585^Δ1 and the molecular nature of both tweek alleles are indicated. tweek¹ harbors a 74 bp deletion (red) and a 6 bp insertion (indicated) and tweek² harbors a splice acceptor mutation before exon 20. RT-PCR on yw eyFLP; FRT40A^iso control, tweek^c01084 and tweek^c01084/Df(2L)Exel8036 using the primers shown in Supplemental Table 1. (D) In situ hybridization of dioxygenin labeled RNA to whole stage 15 embryos using a CG4841 probe revealing labeling in the mid gut (MG), hind gut (HG), brain lobe (BL) and ventral nerve cord (VNC). An independent probe against CG15134 shows an identical labeling pattern (see Figure S3) while sense probes do not show specific labeling.
(E) Lethal stage of tweek mutant combinations. L1-2: animals do not survive beyond the first or second instar larval stage. Pupa or Unc: most animals die during the pharate adult (late pupal) stage. However, very few (<1/2,000) manage to eclose but are severely uncoordinated. −: failure to complement, +: complement.