The molecular mechanisms underlying the retrograde control of presynaptic neurotransmitter release at the NMJ are not well understood. Here we describe the identification of the first known inhibitor of synaptic homeostasis. Identification of the pathways through which this inhibitor act may eventually lead to a greater understanding of the mechanisms that regulate homeostasis.
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.
Neuromuscular Junctions in Drosophila gathers the main contributions that research using the fruit fly Drosophila melanogaster has made in the area of synapse development, synapse physiology, and excitability of muscles and nerve cells. The chapters in this book represent a synthesis of major advances in our understanding of neuronal development and synaptic physiology, which have been obtained using the above approach.This book is directed to the general neuroscience audience: researchers, instructors, graduate students, and advanced undergraduates who are interested in the mechanisms of synapse development and physiology. However, the book will also be a valuable resource for those that use the fruit fly as a model system in their laboratories. Key Features* Synthesizes the genetic approaches used to study synaptic development and function at the neuromuscular junction, using flies as a model system* Covers major recent advances in muscle development, pathfinding, synapse maturation and plasticity, exo- and endocytosis, and ion channel function* Written in clear language that is easily understandable to readers not already familiar with fruit fly research* Includes numerous diagrams and extensive reference lists
