Regulation of synaptic structure and function at the Drosophila neuromuscular junction

Abstract

Neuronal communication requires a spatially organized synaptic apparatus to coordinate neurotransmitter release from synaptic vesicles and activation of postsynaptic receptors. Structural remodeling of synaptic connections can strengthen neuronal communication and synaptic efficacy during development and behavioral plasticity. Here, I describe experimental approaches that have revealed how the actin cytoskeleton participates in transynaptic signaling to control synapse assembly. I also describe my studies on how regulation of endocytic trafficking controls synaptic growth during neuronal development. To identify regulators of synapse assembly, I carried out a large-scale EMS mutagenesis screen of the second chromosome. From this screen I identified a mutation in actin 57B that disrupts synaptic morphology and presynaptic active zone organization. Actin 57B is one of six actin genes in Drosophila and is expressed in body wall muscle during larval development. The isolated allele harbors a point mutation disrupting a highly conserved amino acid present throughout the actin family. Homozygous mutant larvae show impaired alignment and spacing of presynaptic active zones. Additionally, disruption of the organization of the postsynaptic density is observed, with mislocalization of the Spectrin cytoskeleton and the PSD-homolog Disc-Large. Phallodin staining reveals a severe disruption of postsynaptic actin surrounding presynaptic boutons, with the formation of aberrant large actin swirls. Based on these results, we hypothesize that the loss of a synaptic interaction mediated by actin 57B leads to disruption of postsynaptic cytoskeletal organization and dysregulation of signals required to organize presynaptic active zones. Additionally, I present data that provide new insights into the mechanisms controlling synaptic growth signaling during transit through the endocytic pathway. Nervous Wreck (Nwk) is a presynaptic F-BAR/SH3 protein that regulates synaptic growth signaling in Drosophila. Here, I show that Nwk acts through a physical interaction with Sorting Nexin 16 (SNX16). SNX16 promotes synaptic growth signaling by activated BMP receptors, and live imaging in neurons reveals that SNX16-positive early endosomes undergo transient interactions with Nwkcontaining recycling endosomes. We identify an alternative signal termination pathway in the absence of Snx16 that is controlled by ESCRT-mediated internalization of receptors into the endosomal lumen. Our results define a presynaptic trafficking pathway mediated by SNX116, NWK and the ESCRT complex that functions to control synaptic growth signaling at the interface between endosomal compartments. Together, these experiments have expanded our understanding of the molecular mechanisms that control synaptic growth and assembly, highlighting the role of the postsynaptic actin cytoskeleton and the presynaptic endosomal trafficking pathway as key regulators.