A perisynaptic ménage à trois between Dlg, DLin-7, and Metro controls proper organization of Drosophila synaptic junctions

Abstract

Structural plasticity of synaptic junctions is a prerequisite to achieve and modulate connectivity within nervous systems, e.g., during learning and memory formation. It demands adequate backup systems that allow remodeling while retaining sufficient stability to prevent unwanted synaptic disintegration. The strength of submembranous scaffold complexes, which are fundamental to the architecture of synaptic junctions, likely constitutes a crucial determinant of synaptic stability. Postsynaptic density protein-95 (PSD-95)/ Discs-large (Dlg)-like membrane-associated guanylate kinases (DLG-MAGUKs) are principal scaffold proteins at both vertebrate and invertebrate synapses. At Drosophila larval glutamatergic neuromuscular junctions (NMJs) DlgA and DlgS97 exert pleiotropic functions, probably reflecting a few known and a number of yet-unknown binding partners. In this study we have identified Metro, a novel p55/MPP-like Drosophila MAGUK as a major binding partner of perisynaptic DlgS97 at larval NMJs. Based on homotypic LIN-2,-7 (L27) domain interactions, Metro stabilizes junctional DlgS97 in a complex with the highly conserved adaptor protein DLin-7. In a remarkably interdependent manner, Metro and DLin-7 act downstream of DlgS97 to control NMJ expansion and proper establishment of synaptic boutons. Using quantitative 3D-imaging we further demonstrate that the complex controls the size of postsynaptic glutamate receptor fields. Our findings accentuate the importance of perisynaptic scaffold complexes for synaptic stabilization and organization.

Figure 1.

Metro is a neural MPP-like MAGUK. A, Domain organization of Metro. The degree of conservation in human MPP3, MPP4, and MPP7 is listed beneath. Splice variations may affect the PDZ and HOOK domains as indicated. The PEST sequence is conserved in MPP3 and MPP7. A bar indicates the region used for generating antibodies. B, Northern blot analysis on mRNA of 0–14 h and 14–24 h embryos, third-instar larvae, and adults. C, In situ hybridization on stage 11 and stage 13 embryos (lateral view, anterior left) reveals metro mRNA in the developing CNS.

Figure 2.

DlgS97, Metro, and DLin-7 interact in vitro and at NMJs. A–D, Pull-down experiments. Input controls (left lanes) and fractions bound to immobilized GST fusion proteins were analyzed by Western blot analysis using streptavidin-peroxidase conjugates for detection. E, Coimmunoprecipitation of Metro together with DLin-7 from body wall muscle extracts. Preimmune serum was used as a control. F–G, Maximum projections of confocal stacks displaying the localization of Metro relative to DLin-7, HRP epitope (F–F″) and glutamate receptors (G) at NMJs of muscle 12 (F–F″) or 4 (G). Scale bars: 10 μm.

Figure 3.

Loss of metro abolishes DLin-7 at NMJs. A, Exon–intron structure of metro and mapping of mutant alleles. Open and filled boxes represent untranslated and coding regions, respectively. Imprecise excision of the P-element BG02148 yielded hypomorphic alleles (*35, *56) and the null allele *47, a deletion lacking exons 2–5 and part of exon 6. Df(2R)E3363 uncovers metro completely. B, Western blot analysis of body wall extracts from the wild type, metro mutants (*47/Df), muscle-specific RNAi (UAS-metro^dsRNA; Gal4–C57), and overexpression (UAS-metro-B;; Gal4–C57). The same blot was probed for DlgS97 (120 kDa) and Metro (70 kDa). C–E″, NMJs at muscles 6/7 of the wild type, metro mutants, and metro mutants expressing a metro-B transgene in muscles, stained for HRP (C–E), Metro (C′–E′), and DLin-7 (C″–E″). F, DLin-7 appears unaffected at cmg mutant NMJs. G, H, Flag-tagged DLin-7 localized to type I boutons at muscle 12 in the presence (G) but not in the absence (H) of Metro. I–K, Presynaptic DLin-7 visualized at muscles 12/13 upon neuron-specific expression of UAS-metro-B in metro mutants (J); controls lacking the Gal4 activator (I, I′); DLin-7 localizes next to Brp-labeled active zones (K). Scale bars: (in E″, F, I′, and J) C–F, I–J, 40 μm; (in H) G, H, 20 μm; K, 10 μm.

Figure 4.

DlgS97 and spectrin contribute to NMJ localization of Metro and DLin-7. A, B, Western blot analysis of isoform-specific dlg-alleles A-51.1 and S97-flpV next to the conventional allele XI-2. Detection of β-tubulin served as a loading control. Absence of DlgS97 in dlg^S97-flpV mutant body wall extracts was validated by an isoform-specific antiserum (A). Anti-Dlg_PDZ-antibodies reveal the absence of the 97 kDa DlgA isoform in dlg^A-51.1 mutant body walls (B). A scheme illustrates that DlgS97 and an alternative DlgA isoform (DlgA-XL) contribute to the 120 kDa band. C–E, Metro at wild-type, dlg^A-51.1, and dlg^S97-flpV mutant NMJs. F–G′, Disproportionate reduction of DLin-7 relative to total Dlg at dlg^S97-flpV mutant NMJs. H, Normalized fluorescence intensities for DLin-7 and Dlg at wild-type versus mutant NMJs. Each of the plotted values is derived from at least four segmented muscle 6/7 NMJ branches from a single larva. DLin-7 fluorescence levels were as follows: wild type, 100 ± 8.8%; dlg^S97-flpV, 23.7 ± 4.0% (t test: p < 0.001). Dlg fluorescence levels were as follows: wild type, 100 ± 7.4%; dlg^S97-flpV, 73.7 ± 9.7% (t test: p < 0.001); I–J′, Costaining of NMJs lacking DlgS97 and/or postsynaptic spectrin for HRP and DLin-7. Compared with the wild type (F), DLin-7 is hardly affected at spectrin-depleted NMJs heterozygous for dlg^S97-flpV (I′). Combinatorial loss of DlgS97 and spectrin (J, J′) reduces junctional DLin-7 clearly beyond its already diminished level at dlg^S97-flpV mutant NMJs (compare J′ to G). Note that images in F to J′ are derived from samples treated in parallel. All depicted NMJ branches are from muscles 6 or 7 of segment A2. Scale bars: 10 μm.

Figure 5.

Metro is required for proper growth and morphology of NMJs. A, Quantification of Ib boutons on muscles 6/7 of segment A2, normalized to 100,000 μm² of muscle surface area. Wild type, 114.63 ± 18.29 (mean ± SD); metro*⁴⁷, 75.30 ± 20.12; metro*⁴⁷ plus ubiquitous Metro-B, 105.05 ± 19.01. Numbers of scored NMJs are indicated at the bottom of each column. Error bars correspond to SEMs. n.s., No significant difference, p > 0.05; ***, highly significant, p < 0.001 (t test). B, Quantification of Ib bouton projection areas. Each plotted value is a mean value derived from the analysis of boutons at a single NMJ (supplemental Table 1, available at www.jneurosci.org as supplemental material). Mean bouton areas ± SD were as follows: wild type, 4.14 ± 0.38 μm²; metro*⁴⁷, 6.43 ± 1.1 μm²; metro*⁴⁷ plus ubiquitous Metro-B, 4.77 ± 0.67 μm². The statistical test was the nonparametric Kruskal–Wallis test plus Dunn's multiple comparison. n.s., Not significant, p > 0.05; *p < 0.05; ***p < 0.001. n corresponds to the number of NMJs analyzed. B′, Percentage of boutons with projection areas of >10 μm². C, D, anti-HRP-stained NMJ branches of wild-type (C) and metro*⁴⁷ mutant (D) larvae. Asterisks mark striking corpuscles typically associated with mutant NMJs. E–F′, Boutons with impaired differentiation as indicated by the absence of Dlg (E, arrow) and/or by the low abundance of Brp- and GluRIID-immunoreactive spots (F, F′, arrowheads) were found on virtually each metro mutant NMJ. Scale bars: 5 μm.

Figure 6.

GluR fields are enlarged in metro mutants. A, B, Maximum projections of confocal stacks of wild-type (A) and homozygous metro*⁴⁷ (B) NMJ branches stained for GluRIID. A′–B″, Blend mode 3D representations of GluR fields obtained by surface segmentation are shown with volume-rendered HRP labeling (A′, B′) or with spot-detected Brp staining (A″, B″). Images correspond to framed areas in A and B. Note that the HRP staining hides several GluR fields due to the 3D reconstruction. Arrowheads in B′ point to boundaries of closely juxtaposed GluR fields. Occasionally, image processing may lead to inappropriate segmentation of large, irregularly shaped receptor fields, yielding fields that lack adjunct Brp spots (asterisks in B″). Note that each Brp spot is associated with a receptor field. C, Quantification of GluR field sizes. Each of the plotted values is the mean value derived from the analysis of at least 200 receptor fields at a single NMJ and n corresponds to the number of NMJs analyzed. Mean sizes ± SD were as follows: wild type, 1.301 ± 0.114 μm²; dlg^S97-flpV, 1.792 ± 0.133 μm²; metro*⁴⁷, 1.888 ± 0.160 μm²; metro*⁴⁷ plus ubiquitous Metro-B, 1.545 ± 0.154 μm²; metro*⁴⁷/ Df, 1.973 ± 0.462 μm²; dlg^S97-flpV; metro*⁴⁷, 1.704 ± 0.201 μm². n.s., Not significant, p > 0.05; **p < 0.01; ***p < 0.001 (nonparametric Kruskal–Wallis test plus Dunn's multiple comparison). C′, Ratios between the number of Brp spots and the number of receptor fields, determined for the same samples as in C. Mean values are indicated. No significant differences were detected (Kruskal–Wallis test). D–F, Electron micrographs of wild-type (D) and mutant (E, F) type Ib boutons. Arrowheads in D delineate an electron-dense active zone carrying a T-bar. An example of closely juxtaposed electron-dense zones is shown in F with arrows pointing to minimal spacings. Scale bars: (in B) A, B, 5 μm; D–F, 1 μm.

Figure 7.

Basal transmission is normal at metro mutant NMJs. A, Electrophysiological recordings of spontaneous release events. No significant differences were evident between controls (+/Df) and mutants (*47/Df) regarding the frequency (1.25 ± 0.31 vs 1.19 ± 0.10 Hz) and amplitude (−0.91 ± 0.05 vs −0.83 ± 0.03 nA) of mEJCs. B, The amplitudes of evoked responses upon 0.2 Hz stimulation were comparable between controls and mutants (−75.7 ± 6.1 vs −86.3 ± 4.3 nA), as were the number of vesicles released per action potential (quantal content; 84 ± 7 vs 107 ± 8).

Figure 8.

DLin-7 controls NMJs levels of Metro and DlgS97. A–C, Reduction of junctional DlgS97 due to loss of Metro (B) is restored by muscle expression of Metro-B (C). NMJ branches are shown with contours to illustrate type Ib bouton-specific definition of ROIs as a basis for reliable measurement of fluorescence intensities. D, Fluorescence intensities. Each symbol corresponds to the normalized mean value derived from measuring at least two branches of an individual NMJ. Wild type, 100% (±8.6% SD); metro*^47/Df, 56.7 ± 10.6% (ANOVA, p < 0.001); rescue (UAS-metro-B; metro*^47/Df; Gal4–C57), 90.3 ± 10.3% (ANOVA, p > 0.05 vs wild type). E–F′, DLin-7 mutant NMJs exhibit reduced levels of DlgS97 (E, F) and virtually no Metro (E′, F′). G, RT-PCR analysis on RNA from larval body walls confirms the presence and absence of metro transcripts in DLin-7*⁶⁶ and metro*⁴⁷ mutants, respectively. RT-PCR for rp49 served as a control. H, Western blot analysis on body wall extracts from various genotypes as indicated. The same samples were probed with Metro- and DLin-7-specific antisera. Coomassie staining served as a loading control. Note the absence of Metro in DLin-7 mutants, which can be overcome by Gal4–C57-driven expression of UAS-metro-B. I, I′, Overexpressed Metro fails to localize at NMJs in the absence of DLin-7. J, Schematic model depicting the promotional effect of DLin-7 on the binding of Metro to DlgS97. Only the L27 domains are shown. K, Diagrammatic representation of the degree of interdependency between DlgS97, Metro, and DLin-7 in regard to NMJ localization. Scale bars: (in C, F′), A–C, E–F′, 10 μm; (in I′) I, I′, 40 μm.

Figure 9.

DLin-7 mutants show aberrant NMJ organization. A, B, Blend mode 3D representations of NMJ branches from the wild type and DLin-7*⁶⁶ mutants stained for GluRIID and HRP. Note the presence of boutons with little or no GluRIID (arrowheads) and the striking HRP-stained corpuscles (asterisks). C, Quantification of bouton projection areas. Each plotted value is a mean value derived from the analysis of type Ib boutons at a single NMJ. Mean bouton areas ± SD were as follows: wild type, 4.14 ± 0.38 μm²; DLin-7^#66/Df, Gal4–C57, 6.73 ± 0.69 μm²; DLin-7^#66Df plus presynaptic and postsynaptic DLin-7, 4.70 ± 0.83 μm². D, Quantification of GluR field sizes. Each plotted value is a mean value derived from the analysis of at least 200 GluR fields at a single NMJ. Mean sizes ± SD were as follows: wild type, 1.301 ± 0.114 μm²; DLin-7^#66/Df, Gal4–C57, 1.624 ± 0.157 μm²; DLin-7^#66Df plus presynaptic and postsynaptic DLin-7, 1.236 ± 0.217 μm². The statistical test in C and D was the nonparametric Kruskal–Wallis test plus Dunn's multiple comparison. n.s., Not significant, p > 0.05; **p < 0.01; ***p < 0.001. n always corresponds to the number of NMJs analyzed.