Postsynaptic actin regulates active zone spacing and glutamate receptor apposition at the Drosophila neuromuscular junction

Highlights

• An E84K mutation was identified in the spectrin binding domain of Actin 57B.

• Disruption of the postsynaptic cytoskeleton alters active zone organization.

• Loss of the actin–spectrin network disrupts active zone-receptor alignment.

• act^E84K genetically interacts with the neurexin transsynaptic signaling complex.

Abstract

Synaptic communication requires precise alignment of presynaptic active zones with postsynaptic receptors to enable rapid and efficient neurotransmitter release. How transsynaptic signaling between connected partners organizes this synaptic apparatus is poorly understood. To further define the mechanisms that mediate synapse assembly, we carried out a chemical mutagenesis screen in Drosophila to identify mutants defective in the alignment of active zones with postsynaptic glutamate receptor fields at the larval neuromuscular junction. From this screen we identified a mutation in Actin 57B that disrupted synaptic morphology and presynaptic active zone organization. Actin 57B, one of six actin genes in Drosophila, is expressed within the postsynaptic bodywall musculature. The isolated allele, act^E84K, harbors a point mutation in a highly conserved glutamate residue in subdomain 1 that binds members of the Calponin Homology protein family, including spectrin. Homozygous act^E84K mutants show impaired alignment and spacing of presynaptic active zones, as well as defects in apposition of active zones to postsynaptic glutamate receptor fields. act^E84K mutants have disrupted postsynaptic actin networks surrounding presynaptic boutons, with the formation of aberrant actin swirls previously observed following disruption of postsynaptic spectrin. Consistent with a disruption of the postsynaptic actin cytoskeleton, spectrin, adducin and the PSD-95 homolog Discs-Large are all mislocalized in act^E84K mutants. Genetic interactions between act^E84K and neurexin mutants suggest that the postsynaptic actin cytoskeleton may function together with the Neurexin–Neuroligin transsynaptic signaling complex to mediate normal synapse development and presynaptic active zone organization.

Introduction

The presynaptic active zone (AZ) organizes the synaptic release machinery to facilitate neuronal communication (reviewed in Südhof, 2013). The AZ is composed of an electron-dense cytomatrix enriched with specialized proteins that serve as a scaffold for the synaptic vesicle trafficking machinery (Sigrist and Schmitz, 2011). The organization of AZs, including their spacing, density and alignment with postsynaptic receptor fields, is tightly regulated to ensure efficient synaptic transmission. AZ density at neuromuscular junctions (NMJs) remains constant throughout development (Clarke et al., 2012, Meinertzhagen et al., 1998, Nishimune, 2012), indicating tight control over AZ assembly. Transsynaptic cell adhesion complexes are thought to mediate coordinated assembly of pre- and postsynaptic specializations (Dalva et al., 2007, Giagtzoglou et al., 2009, Sun and Xie, 2012), although the signaling pathways underlying AZ organization are largely unknown.

The Drosophila NMJ serves as a model synapse for identifying regulators of AZ biology. Previous genetic screens have revealed several signaling pathways that modulate AZ assembly (Sigrist and Schmitz, 2011). A diverse set of proteins has been identified in these screens, ranging from the endocytosis regulator synaptojanin (Dickman et al., 2006), the synaptic vesicle associated GTPase Rab3 (Graf et al., 2009), the serine threonine kinase Unc-51 (Wairkar et al., 2009) to the postsynaptic spectrin cytoskeleton (Pielage et al., 2006). In addition, proper alignment of the AZ with postsynaptic receptors requires transsynaptic communication provided by the Neurexin–Neuroligin and Teneurin synaptic cell adhesion proteins (Banovic et al., 2010, Li et al., 2007, Mosca et al., 2012).

To identify additional pathways that control synapse organization, we carried out a chemical mutagenesis screen in Drosophila. Using fluorescently-conjugated antibodies against the presynaptic AZ protein Bruchpilot (BRP) (Wagh et al., 2006) and the postsynaptic glutamate receptor subunit III (GluRIII) (Marrus, 2004), we screened for mutants that disrupted AZ size, number, location or apposition to postsynaptic glutamate receptors. From the screen we identified a mutation in Actin 57B, one of six actin genes in Drosophila and the main isoform present in postsynaptic muscles (Fyrberg et al., 1980, Tobin et al., 1980), that disrupted normal AZ organization. Here we describe our characterization of the isolated allele, which results from mutation of glutamate 84 (act^E84K) in subdomain 1 of actin, a region required for spectrin binding. We find that postsynaptic Actin 57B is a key organizer of AZ assembly, linking several protein networks to ensure coordinated pre- and postsynaptic maturation. Postsynaptic F-actin structure is severely disrupted in act^E84K mutants, with abnormal actin swirls replacing the typical uniform structure of the actin-rich postsynaptic domain. In addition, the structure of the postsynaptic density is perturbed, with defects in subsynaptic reticulum (SSR) formation and mislocalization of Discs-Large, spectrin and adducin. The disruption of the postsynaptic cytoskeleton leads to a reduction in AZ density and an increase in unapposed AZs and glutamate receptor clusters. These findings indicate that synaptic interactions altered in act^E84K mutants perturb postsynaptic cytoskeletal structure and disrupt transsynaptic signals required for presynaptic AZ organization.