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A Syd-1 homologue regulates pre- and postsynaptic maturation in Drosophila - PubMed

  • ️Fri Jan 01 2010

A Syd-1 homologue regulates pre- and postsynaptic maturation in Drosophila

David Owald et al. J Cell Biol. 2010.

Abstract

Active zones (AZs) are presynaptic membrane domains mediating synaptic vesicle fusion opposite postsynaptic densities (PSDs). At the Drosophila neuromuscular junction, the ELKS family member Bruchpilot (BRP) is essential for dense body formation and functional maturation of AZs. Using a proteomics approach, we identified Drosophila Syd-1 (DSyd-1) as a BRP binding partner. In vivo imaging shows that DSyd-1 arrives early at nascent AZs together with DLiprin-alpha, and both proteins localize to the AZ edge as the AZ matures. Mutants in dsyd-1 form smaller terminals with fewer release sites, and release less neurotransmitter. The remaining AZs are often large and misshapen, and ectopic, electron-dense accumulations of BRP form in boutons and axons. Furthermore, glutamate receptor content at PSDs increases because of excessive DGluRIIA accumulation. The AZ protein DSyd-1 is needed to properly localize DLiprin-alpha at AZs, and seems to control effective nucleation of newly forming AZs together with DLiprin-alpha. DSyd-1 also organizes trans-synaptic signaling to control maturation of PSD composition independently of DLiprin-alpha.

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Figures

Figure 1.
Figure 1.

Proteomics identify DSyd-1 as physical interactor of BRP. (A) Monoclonal BRPNc82 efficiently precipitates BRP (arrowhead), as seen in this SYPRO red–stained SDS-gel. Among other proteins, DSyd-1 was found to coprecipitate with BRP, as confirmed by MS/MS analysis. (B) Matrix showing yeast two-hybrid assay results confirming a direct physical interaction between BRP and DSyd-1. A C-terminal domain of BRP (aa 1,152–1,740) was positive for interaction with a C-terminal region of DSyd-1 (aa 1,301–1,844). Moreover, a bait N-terminal DSyd-1 (aa 1–400) fragment interacted with both the N-terminal fragment of BRP (aa 1–320) and a C-terminal BRP (aa 1,152–1,740) fragment. (C) Genomic location of dsyd-1 on chromosome arm 3R at 100D2-100D3. dsyd-1–deficient animals were constructed using Drosophila lines carrying transposon-mediated flippase recognition target sites (Parks et al., 2004) that neighbored the dsyd-1 locus (black, dsyd-1ex1.2; gray, dsyd-1ex3.4 in gray). We obtained two deficiencies that were confirmed with genomic PCR. In both cases, the entire dsyd-1 locus (red) was excised, whereas in one case (dsyd-1ex1.2, black line), the 5′ ferrochelatase was affected; and in the other case, the 3′ heph (dsyd-1ex3.4, gray line) locus was affected. Taking these deficiencies in trans eliminates both copies of dsyd-1; however, this leaves one intact copy of each heph and ferrochelatase. (D and E) Behavioral tests demonstrate a requirement for DSyd-1 and less stringent requirement for DLiprin-α in the adult CNS. (D) Walking ability (control: 15.69 ± 0.57 lines, n = 15; dsyd-1: 1.62 ± 0.69 lines, n = 8; dsyd-1rescue: 12.86 ± 0.99 lines, n = 10; dliprin-α: 16.19 ± 0.65 lines, n = 7; control × dsyd-1: P = 0.0001; control × dsyd-1rescue: P = 0.02; control × dliprin-α: P = 0.67; dsyd-1 × dsyd-1rescue: P < 0.0001). (E) Negative geotaxis (control: 8.32 ± 0.37 cm; dsyd-1: 2.92 ± 0.60 cm; dsyd-1rescue: 8.833 ± 0.17 cm; dliprin-α: 8.67 ± 0.15 cm; all: n = 10; control × dsyd-1: P < 0.0001; control × dsyd-1rescue: P = 0.32; control × dliprin-α: P = 0.91; dsyd-1 × dsyd-1rescue: P < 0.0001). Impaired locomotive behavior in dsyd-1 flies is rescued by pan-neural (elav-GAL4) reexpression of the dsyd-1 cDNA. Error bars indicate the SEM. *, P < 0.05; ***, P < 0.005; ns, P > 0.05. (F) A polyclonal α-DSyd-1 antibody recognizes a band at the predicted molecular mass of 195 kD on immunoblots of w1118 control fly head lysate (arrow). This band is missing in dsyd-1 head extracts. Statistics: Mann-Whitney test.

Figure 2.
Figure 2.

DSyd-1 localizes to central synapses. (A) In situ hybridizations show that dsyd-1 is expressed throughout the embryo’s CNS. st., stage. (B) Confocal z projection of adult Drosophila CNS. α-DSyd-1 staining colocalizes with BRPNc82 throughout the brain, but is absent in dsyd-1 animals (C). (D) DSyd-1 localizes opposite to postsynaptic acetylcholine receptors (Dα7GFP) expressed in Kenyon cells at the adult MB calyx. Arrowheads in the inset panels (which show enlarged views) indicate pre- to postsynaptic alignment. Bars: (B and C) 50 µm; (D) 10 µm; (D, insets) 500 nm.

Figure 3.
Figure 3.

DSyd-1 localizes to a subcompartment surrounding the AZ core. (A) Boutons of larval NMJ innervating muscle 6/7. Most DSyd-1 clusters are found associated with BRPNc82 signal, labeling AZs, as seen in high-magnification images (right). (B) There was no DSyd-1 staining at dsyd-1–deficient NMJs. (C) Single confocal slices of junctions expressing DLiprin-αGFP, as described in Fouquet et al. (2009). STED images of α-GFP labelings show DLiprin-αGFP as discrete spots arranged around the AZ core labeled by BRPNc82. (D) Single confocal slices of NMJs stained for endogenous DSyd-1 (STED) and BRPNc82 (confocal). Distinct separable DSyd-1 spots closely resembling DLiprin-α distribution are arranged around the AZ center. (E) Merged images of several aligned planar imaged AZs of moderate size associated with three DLiprin-α or DSyd-1 clusters. The image shows BRPNc82 in confocal resolution, α-GFP–labeled NMJs (for DLiprin-αGFP), or DSyd-1–labeled NMJs imaged with STED. The arrangement of DSyd-1 clusters resembles that of the DLiprin-α clusters. da, distance between single clusters associated with the AZ; db, distance between AZ associated cluster and AZ center; dc, diameter of clusters associated with AZs. (F) Single confocal slices of junctions expressing DLiprin-αGFP. STED images of α-GFP show DLiprin-αGFP as discrete dots arranged around the AZ core labeled by BRPNc82, ranging from one or two dots at small AZs to four or five dots at mature-sized AZs. (G) Triple labeling for DLiprin-αYFP, DSyd-1, and BRP. Bars: (A and B) 2 µm; (A and B, insets) 500 nm; (C and D), 1 µm; (C and D, insets): 250 nm; (E) 250 nm; (F) 250 nm; (G) 500 nm.

Figure 4.
Figure 4.

Comparative analysis of NMJ morphology and function in dsyd-1 and dliprin-α mutant animals. (A) Mean traces (left) of eEJCs at 0.2-Hz nerve stimulation recorded from the larval NMJ at 1 mM extracellular calcium (muscle 6) for controls, dsyd-1, and dsyd-1rescue; and mean eEJC amplitudes (right) for dsyd-1 and control group (control: 99.3 ± 9.6 nA; dsyd-1: 59.2 ± 5.9 nA; both: n = 9, P = 0.01), dsyd-1rescue (dsyd-1: 81.4 ± 4.5 nA, n = 9, P = 0.003; control: P = 0.162) as well as for dliprin-α and the control group (control: −89.4 ± 7.7 nA; dliprin-α: −62.0 ± 4.3 nA; both: n = 7, P = 0.007). Both dliprin-α and dsyd-1 show reduced amplitudes compared with controls. This defect is significantly rescued by reexpressing dsyd-1 cDNA in dsyd-1–deficient animals using a motoneuron-specific driver (ok6-GAL4). (B) Sample traces of mEJCs (right) for control, dsyd-1, and dsyd-1rescue animals. Mean mEJC amplitudes (left) for controls (0.93 ± 0.05 nA, n = 7), dsyd-1 (0.91 ± 0.05 nA, n = 8), and dsyd-1rescue (0.86 ± 0.02 nA, n = 9), as well as dliprin-α (0.83 ± 0.05 nA, n = 7) and control (0.90 ± 0.06 nA, n = 7), are comparable (dsyd-1 × control: P = 0.86; dsyd-1 × dsyd-1rescue: P = 0.54; control × dsyd-1rescue: P = 0.14; control × dliprin-α: P = 0.46). (C) dsyd-1 eEJC amplitudes normalized against mean control eEJC amplitude recorded at 1 mM (0.54 ± 0.07, n = 11) or 0.5 mM (0.51 ± 0.05, n = 8; P = 0.48) extracellular calcium, respectively. (D) Paired pulse experiments with a 10-ms (control: 1.58 ± 0.13, n = 11; dsyd-1: 1.81 ± 0.16, n = 13; P = 0.25) or 30-ms (control: 1.31 ± 0.05, n = 12; dsyd-1: 1.33 ± 0.08, n = 12; P = 0.98) interpulse interval recorded at 0.5 mM extracellular calcium. (E) Projection of confocal stacks of muscles 6 and 7 NMJs, labeled with antibodies recognizing BRP (BRPNc82, green) and HRP (magenta). Bars, 10 µm and 1 µm (insets). (F) Morphological size of dliprin-α and dsyd-1 mutant NMJs was reduced compared with controls. The latter was rescued by motoneuron-specific reexpression of dsyd-1cDNA (control: 1.0 ± 0.04, n = 30; dsyd-1: 0.73 ± 0.06, n = 14; dsyd-1rescue: 0.91 ± 0.08, n = 8; dliprin-α: 0.66 ± 0.04, n = 14; control × dsyd-1: P < 0.01; control × dsyd-1rescue: P > 0.05; control × dliprin-α: P < 0.001; dsyd-1 × dsyd-1rescue: P > 0.05; dsyd-1 × dliprin-α: P > 0.05; dsyd-1rescue × dliprin-α: P > 0.05, one-way analysis of variance [ANOVA]). (G) Number of AZs per NMJ counted via α-BRPNc82 labeling. In both dsyd-1 and dliprin-α mutants, AZ numbers were reduced compared with controls. The reduction seen in dsyd-1 mutants was rescued by presynaptic dsyd-1 cDNA expression (control: 704.6 ± 64.94, n = 14; dsyd-1: 508.6 ± 36.07, n = 14; dsyd-1rescue: 673.1 ± 45.30, n = 10; dliprin-α: 247.3 ± 15.81, n = 8; control × dsyd-1: P = 0.020; control × dsyd-1rescue: P = 0.75; control × dliprin-α: P = 0.0002; dsyd-1 × dsyd-1rescue: P = 0.008). (H) Number of PSDs defined by DGluRIID (not depicted). The results were comparable to those in G (control: 712.7 ± 55.24, n = 20; dsyd-1: 523.5 ± 36.35, n = 13; dsyd-1rescue: 667.9 ± 46.85, n = 8; dliprin-α: 281.1 ± 22.83, n = 7; control × dsyd-1: P = 0.025; control × C: P = 0.86; control × dliprin-α: P = 0.0002; dsyd-1 × dsyd-1rescue: P = 0.047). Statistics: Mann-Whitney test. Error bars indicate the SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.005; ns, P > 0.05.

Figure 5.
Figure 5.

Abnormal BRP clusters in dsyd-1. (A–C) BRP puncta (confocal, left), BRP-donuts (STED, middle), and DGluRIID with BRP donuts (right). (A) The control BRP donut is indicated by arrowheads. (B) AZ size (arrowheads) is affected in dsyd-1. BRP donuts lacking postsynaptic DGluRIID receptors are observed as well (arrows). BRP donuts are frequently interconnected and abnormally shaped (arrowheads). (C) Defects are largely rescued by reexpression of UAS–dsyd-1cDNA. Bar, 1 µm. (A’–C’) Magnified views of A–C. Bar, 250 nm. (D) Quantification shows elevated areas of individual BRPNc82 clusters (control: 0.087 ± 0.002 µm2, n = 298; dsyd-1: 0.105 ± 0.005 µm2, n = 265; dsyd-1rescue: 0.091 ± 0.004 µm2, n = 207; control × dsyd-1: P < 0.01; control × dsyd-1rescue: P > 0.05; dsyd-1 × dsyd-1rescue: P > 0.05). (E) Number of individual BRP clusters per single PSDs (control: 1.49 ± 0.05, n = 297; dsyd-1: 2.14 ± 0.12, n = 265; dsyd-1rescue: 1.79 ± 0.08, n = 207; control × dsyd-1: P < 0.001; control × dsyd-1rescue: P > 0.05; dsyd-1 × dsyd-1rescue: P > 0.05). Statistics: one-way ANOVA. Error bars indicate the SEM. **, P < 0.01; ***, P < 0.005.

Figure 6.
Figure 6.

Abnormal organization of T bars and floating electron-dense material in dsyd-1 mutant animals. (A–B’) Serial sections of misshapen T bars at dsyd-1 (B and B’) AZs in comparison to control T bar (A). The arrowhead indicates filaments emerging from an overgrown T bar (B) in dsyd-1. (B’) Immature small T bar in dsyd-1. Reconstruction: red, T bar material; yellow, SVs; membrane blue, AZ. (C) Ectopic electron-dense material can be found at the edge of the AZ membrane (arrowhead, green in reconstruction); electron-dense material (arrowhead, green in reconstruction) associated with SVs is found proximal to AZs (D). (E) Ectopic BRP immunoreactivity (confocal image, left) and ectopic electron-dense material in the center of a dsyd-1 mutant bouton (right). Ectopic electron-dense material (not depicted) and ectopic BRP immunoreactivity (F, arrowheads) are also found in axonal stretches. Bars: (A–B′) 100 nm; (C and D) 150 nm; (E, left) 1 µm; (E, right) 150 nm; (F) 1 µm.

Figure 7.
Figure 7.

DSyd-1 controls postsynaptic GluR field size and composition. (A–C) Co-labeling of DGluRIID and BRPNc82 for control (A), dsyd-1 mutant (B), and presynaptically rescued (C) NMJs. Individual PSDs are indicated by arrowheads. (D–F) Co-labeling of DGluRIIA and DGluRIIB for control (D), dsyd-1 mutant (E), and presynaptically rescued (F) NMJs. (G) Integrated DGluRIID signal (control: 32.25 ± 0.67 au, n = 1,314; dsyd-1: 74.86 ± 2.98 au, n = 335; dsyd-1rescue pre: 46.71 ± 1.60 au, n = 515; dsyd-1rescue post: 81.25 ± 3.54 au, n = 344; control × dsyd-1: P < 0.001; control × dsyd-1rescue pre: P < 0.001; control × dsyd-1rescue post: P < 0.001; dsyd-1 × dsyd-1rescue pre: P < 0.001; dsyd-1 × dsyd-1rescue post: P > 0.05; dsyd-1rescue pre × dsyd-1rescue post: P < 0.001). (H) Integrated DGluRIIA signal (control: 33.88 ± 0.66 au, n = 1,064; dsyd-1: 66.85 ± 2.09 au, n = 667; dsyd-1rescue: 36.31 ± 0.87 au, n = 830; control × dsyd-1: P < 0.001; control × dsyd-1rescue: P > 0.05; dsyd-1 × dsyd-1rescue: P < 0.001). (I) Integrated DGluRIIB signal (E, control: 46.40 ± 0.99 au, n = 934; dsyd-1: 23.85 ± 0.60 au, n = 783; dsyd-1rescue: 35.46 ± 0.89 au, n = 770; control × dsyd-1: P < 0.001; control × dsyd-1rescue: P < 0.001; dsyd-1 × dsyd-1rescue: P < 0.001) size in dsyd-1 mutants. (J) GluR field composition (control: 0.89 ± 0.06, n = 7; dsyd-1: 1.99 ± 0.19, n = 8; dsyd-1rescue: 1.24 ± 0.08, n = 6; control × dsyd-1: P < 0.001; control × dsyd-1rescue: P > 0.05; dsyd-1 × dsyd-1rescue: P < 0.01). Statistics: one-way ANOVA. Error bars indicate the SEM. **, P < 0.01; ***, P < 0.005; ns, P > 0.05. Bars: (A–C) 1 µm; (D–F) 2 µm.

Figure 8.
Figure 8.

Embryonic dsyd-1 phenotypes. (A) High-pressure freeze/freeze substitution–prepared NMJ synapse of control, dsyd-1, dliprin-α, and dliprin-α; dsyd-1 double mutant embryos. All genotypes still form electron-dense projections (T bars) at the AZ. (B) Immunostaining of a region comprising muscles 6/7, 12/13, and 4 in late embryos of control, dsyd-1, dliprin-α, and dliprin-α; dsyd-1. Staining: HRP, BRP, and DGluRIID. (B, middle and bottom) Magnifications showing single synapses, with arrowheads denoting BRP (middle) and DGluRIID (bottom) puncta in the indicated mutants. (C) Quantification of BRP and DGluRIID signals at embryonic synapses. BRP signal in dsyd-1 single mutants is significantly increased compared with control, dliprin-α, and dliprin-α; dsyd-1 double mutants. DGluRIID is increased to a similar extent in dsyd-1 and dliprin-α; dsyd-1 double mutants compared with control and dliprin-α mutant animals. Statistics for BRP were as follows. Control: 1.07 ± 0.027, n = 735; dsyd-1: 2.75 ± 0.11, n = 457; dliprin-α: 1.41 ± 0.079, n = 183; dliprin-α; dsyd-1: 1.51 ± 0.054, n = 446; control × dsyd-1: P < 0.001; control × dliprin-α: P < 0.05; control × dliprin-α; dsyd-1: P < 0.001; dsyd-1 × dliprin-α: P < 0.001; dsyd-1 × dliprin-α; dsyd-1: P < 0.001; dliprin-α × dliprin-α; dsyd-1: P > 0.05. Statistics for DGluRIID were as follows. Control: 1.06 ± 0.029, n = 765; dsyd-1: 3.08 ± 0.10, n = 541; dliprin-α: 1.59 ± 0.080, n = 218; dliprin-α; dsyd-1: 2.90 ± 0.088, n = 612; control × dsyd-1: P < 0.001; control × dliprin-α: P < 0.001; control × dliprin-α; dsyd-1: P < 0.001; dsyd-1 × dliprin-α: P < 0.001; dsyd-1 × dliprin-α; dsyd-1: P > 0.05; dliprin-α × dliprin-α, dsyd-1: P < 0.001. Statistics: one-way ANOVA. Error bars indicate the SEM. Bars: (A) 70 nm; (B, top) 2 µm; (B, middle and bottom) 1 µm.

Figure 9.
Figure 9.

DSyd-1 accumulates early during AZ assembly. Confocal stacks of sequentially in vivo imaged NMJs (muscle 26), Δt = 12 h. NMJs coexpressing GFPDSyd-1 and BRP-shortmStraw (A), and DLiprin-αGFP and mStrawDSyd-1 (B). (A) DSyd-1 preceded BRP (arrows and arrowheads) at 65% of the newly forming AZs, and BRP preceded DSyd-1 at 0%. The situation was not resolved at 35% (n = 37). (B) DLiprin-α and DSyd-1 accumulate in close temporal proximity (arrows and arrowheads): DLiprin-α preceded DSyd-1 at 26% of newly forming AZs, and DSyd-1 preceded DLiprin-α at 6%. The situation was not resolved at 68% (n = 35). Bars: (A and B) 4 µm; (A and B, insets) 500 nm.

Figure 10.
Figure 10.

Defective DLiprin-α localization in dsyd-1 mutants. (A–C) DLiprin-αGFP/BRP-shortmStraw co-imaging in control (A), dsyd-1 (B), and dsyd-1rescue (C) are shown. The localization of DLiprin-α is changed at dsyd-1 mutant NMJs, but is rescued by reexpression of UAS–dsyd-1cDNA in motoneurons. Bars, 2 µm and 500 nm (insets). Arrowheads indicate AZs marked by BRP and arrows indicate ectopic DLiprin-α in dsyd-1 mutants. (D–F) DSyd-1 localizes to AZs in control (D), dliprin-α (E), and brp (F) animals. (G) Model of AZ assembly. Yellow arrow, DSyd-1 regulates DLiprin-α early in assembly; green arrow, DSyd-1 regulates GluR field size; gray arrow, DSyd-1 binds BRP and regulates BRP supply. Bars: (A, top): 2 µm; (A, bottom) 500 nm; (F) 2 µm.

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