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

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, actE84K, 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 actE84K mutants show impaired alignment and spacing of presynaptic active zones, as well as defects in apposition of active zones to postsynaptic glutamate receptor fields. actE84K 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 Disc-Large are all mislocalized in actE84K mutants. Genetic interactions between actE84K 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.

Transmission, Development, and Plasticity of Synapses

“…Consistent with this observation, AZ/GluR size and spacing defects are also seen with disruption of the postsynaptic actin cytoskeleton, as revealed by knockdown of ⍺-or b-spectrin in the muscle (Pielage et al 2006), and analysis of a point mutation in the spectrin-binding domain of the muscle-specific actin gene Actin57B (Blunk et al 2014). These mutants also have severely compromised SSRs.…”

“…These mutants also have severely compromised SSRs. Genetic interaction experiments support cooperation between actin57B, nrx-1, and nlg-1 (Blunk et al 2014). Other mutations affecting the postsynaptic actin cytoskeleton result in a mild increase in GluR cluster size without any reported effects on AZ size or spacing.…”

“…In recent years a number of groups have screened for mutations affecting synaptic organization using antibodies that recognize Brp and GluRs (Graf et al 2009;Wairkar et al 2009;Valakh et al 2012;Blunk et al 2014). These and other studies have defined several molecular pathways that regulate distinct and separable features such as the size, density, and apposition of individual synapses.…”

“…These irregularities of synaptic ultrastructure are typically accompanied by defects in AZ spacing, where multiple Brp puncta cluster opposite single enlarged or fused GluRs. Mutations in several synaptic genes exhibit variations of this phenotype, including genes encoding the CAZ components syd-1 and Liprin-⍺ (Kaufmann et al 2002;Fouquet et al 2009;Owald et al 2010), transsynaptic adhesion genes nrx-1, nlg-1, and the teneurins ten-m and ten-a (Li et al 2007;Banovic et al 2010;Owald et al 2012), and components of the postsynaptic actin cytoskeleton (Pielage et al 2006;Blunk et al 2014). There is evidence of cooperation between many of these genes, which begins to provide a picture of how synaptic organization is coordinated across the synaptic cleft.…”

Mechanisms controlling neuromuscular junction stability

“…It has recently been proposed in Drosophila that a post-synaptic actin cytoskeleton may function together with the Neurexin-Neuroligin transsynaptic signaling complex to mediate normal synapse development and pre-synaptic active zone organization [34,35]. Severe synapse assembly deficits are found in Drosophila melanogaster neurexin (Nrx-1, dnrx) and also neuroligin (Nlg1, dnlg1) mutant [36].…”