Postsynaptic actin regulates active zone spacing and glutamate receptor apposition at the Drosophila neuromuscular junction
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 (actE84K) 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 actE84K 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 actE84K mutants perturb postsynaptic cytoskeletal structure and disrupt transsynaptic signals required for presynaptic AZ organization.
Section snippets
EMS mutagenesis screen for regulators of synaptic growth and organization
The Drosophila NMJ provides a powerful model system to characterize synaptic organization. During its life cycle, Drosophila undergoes 3 larval stages marked by a significant increase in muscle size. To ensure efficient muscle contraction, the NMJ arbor expands ~ 10 fold during development, adding new synaptic boutons and increasing the number of AZs through an activity-dependent process (Stewart et al., 1996, Zito et al., 1999). To identify regulators of AZ organization and synapse formation,
Discussion
Using an unbiased genetic screen for regulators of synaptic growth and organization in Drosophila, we isolated a mutation in Actin 57B that revealed a role for the postsynaptic cytoskeleton in regulating presynaptic AZ density and alignment with postsynaptic glutamate receptor fields. In actE84K mutant animals, the organization of the postsynaptic compartment is severely disrupted, with prominent F-actin swirls and mislocalization of spectrin, adducin and DLG. In addition to its critical role
Genetic screen and Drosophila stocks
Five rounds of ethyl methanesulfonate (EMS) mutagenesis were performed, with 500 isogenized control CS males fed 25 mM EMS for each round. Mutagenized males were crossed to virgins of the second chromosome balancer ln(2LR)Gla, wgGla-1/CyO,GFP. Single male progeny were crossed to ln(2LR)Gla, wgGla-1/CyO,GFP, and stocks of the mutagenized chromosome balanced with CyO,GFP were established. Homozygous mutant non-GFP 3rd instar larvae from each stock were dissected and stained using anti-Bruchpilot
Acknowledgments
This work was supported by NIH grant NS40296 (J.T.L.).
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