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. 2009 Jul 30;63(2):203-15.
doi: 10.1016/j.neuron.2009.06.017.

Tweek, an evolutionarily conserved protein, is required for synaptic vesicle recycling

Patrik Verstreken  1 , Tomoko Ohyama, Claire Haueter, Ron L P Habets, Yong Q Lin, Laura E Swan, Cindy V Ly, Koen J T Venken, Pietro De Camilli, Hugo J Bellen
Affiliations

Tweek, an evolutionarily conserved protein, is required for synaptic vesicle recycling

Patrik Verstreken et al. Neuron. .

Abstract

Synaptic vesicle endocytosis is critical for maintaining synaptic communication during intense stimulation. Here we describe Tweek, a conserved protein that is required for synaptic vesicle recycling. tweek mutants show reduced FM1-43 uptake, cannot maintain release during intense stimulation, and harbor larger than normal synaptic vesicles, implicating it in vesicle recycling at the synapse. Interestingly, the levels of a fluorescent PI(4,5)P(2) reporter are reduced at tweek mutant synapses, and the probe is aberrantly localized during stimulation. In addition, various endocytic adaptors known to bind PI(4,5)P(2) are mislocalized and the defects in FM1-43 dye uptake and adaptor localization are partially suppressed by removing one copy of the phosphoinositide phosphatase synaptojanin, suggesting a role for Tweek in maintaining proper phosphoinositide levels at synapses. Our data implicate Tweek in regulating synaptic vesicle recycling via an action mediated at least in part by the regulation of PI(4,5)P(2) levels or availability at the synapse.

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Figures

Figure 1
Figure 1. tweek mutant photoreceptors show synaptic defects
(A) Electroretinograms of controls (yw eyFLP; P{y+} FRT40A / l(2)cl-2L P{w+} FRT40A), tweek mutants (yw eyFLP; tweek1 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.
Figure 2
Figure 2. Characterization of tweek mutants
(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 tweek1 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 EY02585Δ1 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; FRT40Aiso control, tweekc01084 and tweekc01084/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.
Figure 3
Figure 3. Synaptic vesicle endocytosis is impaired in tweek mutants
(A) Average EJP amplitude recorded in 1 mM Ca2+ in controls, tweek1/tweek2, tweek2/Df and tweekc01084/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, tweekc01084/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), tweekc01084 / tweek2 mutants (eyFLP; tweek2 P{y+} FRT40A / tweekc01084), 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.
Figure 4
Figure 4. Vesicle size and number are altered in tweek mutants
(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.
Figure 5
Figure 5. Quantal size is increased in tweek mutants
(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 tweekc01084/ tweek2: yw eyFLP; tweek2 P{y+} FRT40A / tweekc01084). (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).
Figure 6
Figure 6. Endocytic adaptor proteins are destabilized at tweek mutant boutons
(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.
Figure 7
Figure 7. Synaptic PI(4,5)P2 levels are reduced in tweek mutants
(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-PHMUT-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.
Figure 8
Figure 8. Removal of a single mutant copy of synaptojanin suppresses endocytic defects in tweek
(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.
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References

    1. Achiriloaie M, Barylko B, Albanesi JP. Essential role of the dynamin pleckstrin homology domain in receptor-mediated endocytosis. Mol Cell Biol. 1999;19:1410–1415. - PMC - PubMed
    1. Babcock MC, Stowers RS, Leither J, Goodman CS, Pallanck LJ. A genetic screen for synaptic transmission mutants mapping to the right arm of chromosome 3 in Drosophila. Genetics. 2003;165:171–183. - PMC - PubMed
    1. Bai J, Tucker WC, Chapman ER. PIP2 increases the speed of response of synaptotagmin and steers its membrane-penetration activity toward the plasma membrane. Nat Struct Mol Biol. 2004;11:36–44. - PubMed
    1. Bellen HJ, Levis RW, Liao G, He Y, Carlson JW, Tsang G, Evans-Holm M, Hiesinger PR, Schulze KL, Rubin GM, et al. The BDGP gene disruption project: single transposon insertions associated with 40% of Drosophila genes. Genetics. 2004;167:761–781. - PMC - PubMed
    1. Betz WJ, Bewick GS. Optical analysis of synaptic vesicle recycling at the frog neuromuscular junction. Science. 1992;255:200–203. - PubMed

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