Drosophila Neto is essential for clustering glutamate receptors at the neuromuscular junction

Abstract

Neurotransmitter receptor recruitment at postsynaptic specializations is key in synaptogenesis, since this step confers functionality to the nascent synapse. The Drosophila neuromuscular junction (NMJ) is a glutamatergic synapse, similar in composition and function to mammalian central synapses. Various mechanisms regulating the extent of postsynaptic ionotropic glutamate receptor (iGluR) clustering have been described, but none are known to be essential for the initial localization and clustering of iGluRs at postsynaptic densities (PSDs). We identified and characterized the Drosophila neto (neuropilin and tolloid-like) as an essential gene required for clustering of iGluRs at the NMJ. Neto colocalizes with the iGluRs at the PSDs in puncta juxtaposing the active zones. neto loss-of-function phenotypes parallel the loss-of-function defects described for iGluRs. The defects in neto mutants are effectively rescued by muscle-specific expression of neto transgenes. Neto clustering at the Drosophila NMJ coincides with and is dependent on iGluRs. Our studies reveal that Drosophila Neto is a novel, essential component of the iGluR complexes and is required for iGluR clustering, organization of PSDs, and synapse functionality.

Figure 1.

Drosophila Neto is essential for muscle function. (A) Alignment of vertebrate Neto1 and Neto2 and Drosophila Neto indicates their similar domain organization. All Neto proteins contain a signal peptide (S), followed by two extracellular protein–protein interaction domains (CUB) and an LDLa motif. The percentages of similarity/identity are as follows: CUB1 domain, 82%/64% between mouse Neto1 and Neto-2, and 54%/41% between fly and mouse Netos; CUB2 domain, 87%/72% and 41%/21%; LDLa, 89%/83% and 50%/31%. The intracellular domain is less conserved (53%/39% and 24%/9%). (B) Schematic view of the neto locus and the small deletions generated. The predicted neto locus has 11 exons spanning a 73-kb region. Small deficiencies were generated by imprecise excisions of transposomal elements located within the neto locus (MB05569 and MB04917). (C) Climbing index in flies of indicated genotypes. Flies were placed at the bottom of test vials and monitored for their climbing abilities within 10 sec. Most neto¹⁰⁹ escapers remained at the bottom of the test vials. (D) Percent of rescue of neto³⁶ embryos into the larval stages by tissue-specific neto expression. The embryonic lethality of neto³⁶ mutants was rescued by ubiquitous expression of the UAS-neto-V5 transgene (da-Gal4) and by expression in muscles (24B-Gal4 and G14-Gal4), but not by expression of Neto in neurons (elav-Gal4) or trachea (btl-Gal4).

Figure 2.

Neto localizes to NMJ. (A–C) Neto localizes in distinct puncta at the NMJ of third instar larvae (A) (detail in A′), late embryos (B), and first instar (C). The anti-HRP antibody (red) labels all motor neuron arbors. The anti-Neto antibody (green) labels predominantly the type I boutons at all NMJ synapses during embryonic and larval stages of development. (Arrow) Type I boutons; (arrowhead) type II boutons. (D,E) Synaptic localization of Neto is disrupted in neto¹⁰⁹ third instar larvae (D), but is restored by a duplication covering the neto locus (E). (D′,E′) Details of sections boxed in D and E. Representative large fields of muscles 6/7 and 12/13 from the indicated genotypes are compared. (F) Muscle-expressed Neto-V5 (green) localizes at the synapses and is distributed in puncta concentrated near the motor neuron arbor (labeled with anti-HRP in red). Excess Neto-V5 accumulates on the muscle membrane in a hollow surrounding the boutons, likely the SSR membrane folds (detail in F′), and in a striped pattern throughout the muscle fiber.

Figure 3.

Postsynaptic localization of Neto. (A–D) Neto-positive puncta are surrounded by Dlg (A) and colocalize with the iGluRs at the PSDs GluRIIC (B), GluRIIA (C), and GluRIIB (D). (E) Neto-positive puncta juxtapose the Brp-labeled active zones, where the neurotransmitter vesicles are released. Confocal stacks of NMJ 6/7 of abdominal segment A2 are shown in all panels, except for the bouton details, which contain very few sections through the boxed boutons. Bar, 10 μm.

Figure 4.

Neurotransmission in neto mutants can be rescued by muscle expression of Neto. (A) Representative traces of spontaneous mEJPs from wild-type larvae (wt), neto¹⁰⁹ larvae, and neto¹⁰⁹/neto³⁶ larvae rescued with duplication. Note the reduction in both mini frequency and amplitude in neto¹⁰⁹ larval NMJs. The muscle resting potential and input resistance are not affected by the neto mutations. (B,C) Bar graph presentation of mean values for mEJP from wild-type larvae (black), neto¹⁰⁹ larvae (red), and neto¹⁰⁹/neto³⁶ larvae rescued with duplication (blue). (B) mEJP frequency. (C) mEJP amplitude. (*) P < 0.001. Error bars denote ±SEM. (D) Representative traces of evoked EJPs from the indicated genotypes. The Ca²⁺ concentration was 0.8 mM in HL-3 saline. (E,F) Bar graph presentation of mean values for EJP amplitude (E) and quantal content (F) from wild-type larvae (black), neto¹⁰⁹ larvae (red), and neto¹⁰⁹/neto³⁶ larvae rescued with duplication (blue). (**) P < 0.05.

Figure 5.

iGluRs do not cluster in the absence of Neto. (A) GluRIIA clustering in embryos of indicated genotypes at 21 h AEL. The GluRIIA receptor subunit (green) clusters in neto³⁶/+ heterozygous (left) but not in neto³⁶/Y hemizygous (middle) embryos. (Right) The GluRIIA clustering is restored in neto³⁶/Y mutant animals when Neto is provided in the muscle. The anti-HRP antibody (blue) labels all motor neuron arbors. (B) Despite the absence of the iGluRs and Neto at the synapses, neto³⁶/Y hemizygous embryos (shown in the middle panel) still show accumulations of the PSD marker PAK (green) opposite to presynaptic release sites labeled with anti-Brp (red). Clustering of Brp and Pak are similar for all genotypes tested: neto³⁶/+ heterozygous (left), neto³⁶/Y hemizygous (middle), and neto³⁶/Y hemizygous animals rescued by muscle expression of Neto-V5 (right).

Figure 6.

Postsynaptic localization of iGluRs is impaired at reduced Neto levels. (A) GluRIIA and GluRIIC immunoreactivites are shifted from postsynaptic clusters in wild type (wt) to mostly extrajunctional locations in neto¹⁰⁹ third instar larvae. (B) GluRIIB postsynaptic clusters are also disrupted in neto¹⁰⁹ larvae and appear shifted from junctional to extrajunctional locations. The few remaining synaptic GluRIIB puncta (green) are always accompanied by Neto puncta (red) at the motor neuron arbor (blue). (C) Western blot comparison of GluRIIB protein levels in wild-type and neto¹⁰⁹ larval muscle. α-Tubulin was used as a loading control. (D) The synaptic accumulation of PAK (green) is diminished in neto¹⁰⁹ larvae as compared with the wild type, but the postsynaptic PAK-positive puncta mostly colocalize with the Neto-positive puncta (red). (E) The intensity and distribution of the presynaptic active zone marker Brp appear to be normal in neto¹⁰⁹ larvae versus wild type. The intensities and numbers of Brp puncta per bouton are largely similar in wild type and neto¹⁰⁹. (Right) Note the reduced size of Neto-positive puncta. The Neto-positive clusters are still juxtaposing Brp-positive active zones in neto¹⁰⁹ larvae. Representative fields at muscles 6/7 of abdominal segment A2 from the indicated genotypes are compared. Bar, 10 μm.

Figure 7.

NMJ synapse development is altered at reduced Neto levels. (A) Postsynaptic Dlg accumulation is shifted from junctional to extrajunctional location in neto¹⁰⁹ larvae as compared with wild type (wt) (details in insets). Note the reduced Dlg staining at the type Ib boutons (details in the last column) and the absence of Dlg staining at the type Is boutons. (Bottom panels) Synaptic accumulation of Dlg is restored by a duplication covering the neto locus. Representative fields at muscles 6/7 of abdominal segment A2 from the indicated genotypes are compared. (B) Western blot comparison of Dlg protein levels in wild-type and neto¹⁰⁹ larval muscle. α-Tubulin was used as a loading control. (C) Electron micrographs of synaptic boutons from wild-type (left) and neto¹⁰⁹ (right) third instar larvae. SSR, T bars (arrows), and PSDs (brackets) are indicated. The SSR is densely packed around wild-type type Ib boutons, but is sparse in neto¹⁰⁹ mutants. At wild-type synapses, T bars are juxtaposed by electron-dense structures in the synaptic cleft and postsynaptic domain. In contrast, the neto¹⁰⁹ mutant synapses display a loss of electron-dense membrane domains (detail). The pre- and postsynaptic membranes are sinuous and appear disorganized in neto¹⁰⁹ mutants as compared with the tight and close apposition of wild-type synaptic membranes. Bar, 1 μm; in details, 0.1 μm. (D) Percent of adults emerging from neto³⁶-null embryos after a pulse of Neto expression. Control (neto³⁶/+;G14/+) or rescued (neto³⁶/Y;G14>UASg-Neto-GFP) animals were reared at 25°C during the embryo stages. The first instar larvae were kept at the indicated temperatures, and the percentages of adults emerging were plotted. When reared at 18°C, <20% adults hatched. Most of the animals that received a pulse of Neto expression only during embryogenesis died during larval and pupal stages. In contrast, at 25°C, most of the rescued animals lived to adulthood.

Figure 8.

Neto clustering at the NMJ requires iGluRs. (A) Timing of Neto clustering at the NMJ. Neto-GFP was expressed in the muscle (using the muscle-specific promoter 24B-Gal4), and the GFP signal was followed in live embryos. Neto-GFP begins to form clusters at the embryonic NMJ as early as 14 h AEL. Yellow brackets mark the future NMJ. (B) Neto clustering in embryos of indicated genotypes at 21 h AEL. Similar to GluRIIA clusters (green), Neto-positive puncta (red) accumulated postsynaptically in gluRIID heterozygous (left) but not in gluRIID homozygous (right) embryos. The anti-HRP antibody (blue) labels all motor neuron arbors. (C) Coimmunoprecipitation of GluRIIB with Neto-V5 from third instar larvae muscles. Soluble muscle fractions were extracted from rescued larvae (neto³⁶;24B>neto-V5) (left panel) or wild-type control (right panel), immunoprecipitated with anti-V5, and analyzed by Western. (Lane 1) Input, 5% of total extract. (Lane 2) Pulled-down fraction. (Lane 3) Unbound fraction. (*) Truncated form of Neto-V5.